Patent application title: Methods, Compositions and Kits for Use in Prognosis, Characterization and Treatment of Cancer
Inventors:
Chin-Yo Lin (Singapore, SG)
Edison T. Liu (Singapore, SG)
Assignees:
Agency For Science, Technology and Research
IPC8 Class: AC12Q168FI
USPC Class:
435 6
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid
Publication date: 2010-10-14
Patent application number: 20100261165
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Patent application title: Methods, Compositions and Kits for Use in Prognosis, Characterization and Treatment of Cancer
Inventors:
Chin-Yo Lin
Edison T. Liu
Agents:
EDELL, SHAPIRO & FINNAN, LLC
Assignees:
Origin: ROCKVILLE, MD US
IPC8 Class: AC12Q168FI
USPC Class:
Publication date: 10/14/2010
Patent application number: 20100261165
Abstract:
The present invention provides methods, compositions and kits for the
prognosis, characterization and treatment of cancers. The methods,
compositions and kits of the invention comprise a set of markers whose
expression is correlated with estrogen receptor beta function. Therefore,
the methods, compositions and kits of the invention find particular use
in endocrine-related cancers such as breast cancers.Claims:
1. A method of assessing ER-.beta. function, the method comprising:(i)
determining the level of at least one marker selected from the group
consisting of CDC2, CDC6, DNA2L and CKS2; and(ii) using the level of the
at least one said marker as an indication of ER-.beta. function.
2. A method for cancer prognosis, the method comprising:(i) determining the level of ER-.beta. in an ER-.alpha. positive sample of a patient; and(ii) using the level of ER-.beta. in making a disease prognosis for the patient.
3. A method for cancer prognosis according to claim 2, wherein the level of ER-.beta. and at least one marker selected from the group consisting of CDC2, CDC6, DNA2L and CKS2 is determined in the ER-.alpha. positive sample of a patient.
4. A method of selecting a treatment for a cancer patient, the method comprising:(i) determining the level of ER-.beta. in an ER-.alpha. positive sample of the patient; and(ii) using the level of ER-.beta. in selecting an appropriate treatment for the patient.
5. A method of selecting a treatment for a cancer patient according to claim 4, wherein the level of ER-.beta. and at least one marker selected from the group consisting of CDC2, CDC6, DNA2L and CKS2 is determined in the ER-.alpha. positive sample of the patient.
6. A method of characterizing a cancerous cell or a cell suspected of being cancerous, the method comprising:(i) determining the level of ER-.beta.; and(ii) using the level of ER-.beta. in characterizing the cell(s);wherein the cell(s) are ER-.alpha. positive.
7. A method of characterizing a cancerous cell or a cell suspected of being cancerous according to claim 6, wherein the level of ER-.beta. and at least one marker selected from the group consisting of CDC2, CDC6, DNA2L and CKS2 is determined in the ER-.alpha. positive cell(s).
8. A method according to any one of claims 1 to 7 wherein the method comprises determining the level of at least two or three markers selected from the group consisting of CDC2, CDC6, DNA2L and CKS2.
9. A method according to any one of claims 1 to 7 wherein the method comprises determining the level of CDC2, CDC6, DNA2L and CKS2.
10. A method according to any one of claims 2 to 7 wherein the cancer is selected from the group consisting of breast cancer, prostate cancer and colon cancer.
11. A method according to any one of claims 1 to 7 wherein the method further comprises determining the level of a further marker wherein the further marker is not selected from the group consisting of CDC2, CDC6, DNA2L and CKS2.
12. A method according to claim 11 wherein the further marker is selected from Table 4.
13. A method according to any one of claims 2 to 7 wherein the cancer has been treated with adjuvant endocrine therapy.
14. A method according to claim 1 wherein the method is used for: (i) assessing treatment efficacy or cancer progression or regression; (ii) identifying a compound useful for the treatment of cancer; or (iii) assessing the carcinogenic potential of a test compound.
15. An array for assessing ER-.beta. function comprising probes, wherein the probes are configured to determine the level of at least one marker selected from the group consisting of CDC2, CDC6, DNA2L and CKS2, and wherein the determined level of the at least one marker is used to assess ER-.beta. function.
16. An array according to claim 15 wherein the array comprises probes for determining the level of CDC2, CDC6, DNA2L and CKS2.
17. An array according to claim 15 wherein the probes are bound to nucleic acids or proteins derived from a sample which is suspected of being cancerous or which is known to be cancerous.
18. A kit for assessing ER-.beta. function comprising a reagent capable of determining the level of at least one marker selected from the group consisting of CDC2, CDC6, DNA2L and CKS2, and wherein the determined level of the at least one marker is used to assess ER-.beta. function.
19. A kit according to claim 18 comprising reagents for determining the level of CDC2, CDC6, DNA2L and CKS2.
Description:
[0001]All documents cited herein are incorporated by reference in their
entirety for all purposes.
FIELD OF THE INVENTION
[0002]The present invention relates 46 molecular genetic markers for prognosis, characterization and treatment of cancer, in particular breast cancer. In particular, the present invention relates to gene expression profile-based signatures for prognosis, characterization and treatment of cancer.
BACKGROUND OF THE INVENTION
[0003]Breast cancer is thought to be the leading cause of death in women between the ages of 40 and 55. An estimated one million women worldwide are thought to be afflicted with some stage of breast cancer. Men are not immune to breast cancer either. Statistics have shown that if detected early, the five-year breast cancer survival rate exceeds 95%. Thus, considerable emphasis has, been placed on early detection of cellular transformation and tumor formation in breast tissue.
[0004]A marker-based approach to tumor identification and characterization promises improved prognosis and diagnosis of breast cancers. Traditionally, breast cancer diagnosis has relied on histopathological examination of the tumor. In addition to diagnosis of cancers, histopathological examinations also provide information about prognosis and selection of treatment regimens. Clinical parameters such as tumor size, tumour grade, the age of the individual and lymph node metastasis may also aid in prognosis. In addition to histopathological examination, diagnosis and/or prognosis may be determined by imaging methods such as mammograms or other X-ray imaging methods. All these techniques suffer from the drawbacks of cost, subjective interpretation and health risks to the individual.
[0005]Breast cancer is a hormone-dependent cancer and knowledge of whether a tumor is positive or negative for the presence of estrogen receptors (e.g. estrogen receptor α, (ER-α)) is used for prognosis and patient selection for anti-hormonal therapy.
[0006]A second isoform of estrogen receptor (ER) called ER-β, may influence tumor progression in ways different from those mediated by the ER-α isoform (37).
[0007]Estrogens are involved in a number of vertebrate developmental and physiological processes and have been implicated in certain types of endocrine-related cancers (1-4). Hormone response in target tissues is mediated by nuclear receptors that function as ligand-dependent transcription factors. Receptor function is further modulated by post-translational modifications and interactions with other nuclear proteins. Originally, only one type of estrogen receptor (ER) was thought to be involved in hormone signaling. However, a second ER, termed ER-β was subsequently discovered, adding another dimension of complexity to the regulation of hormone response. The original receptor was renamed ER-α (5).
[0008]ER-α and ER-β share 55% identity in their ligand-binding domains and approximately 97% similarity in the DNA-binding domains (DBD). Both ERs bind estradiol with high affinity, but vary in their ability to bind other natural and synthetic ligands and the types of response elicited upon ligand binding (6-8). Reflecting the high degree of similarity in their DBDs, both receptors interact with the same conserved estrogen response element (ERE; 5'-GGTCAnnnTGACC-3') (SEQ ID No: 11) as either homodimers or α/β heterodimers (9-11). Tissue-specific expression and co-expression of receptor subtypes suggest that ER homodimers and heterodimers may mediate distinct hormone responses (12-15). Moreover, the discovery of ERβ variants with different structural and functional characteristics and tissue distribution further highlighted the potential complexity of the interactions between ERs and the mechanisms by which estrogen response is modulated (16-20).
[0009]The predominant impact of estrogen receptor activation appears to be alterations in the transcriptional activity and expression profiles of target genes. A number of genes, including trefoil factor 1/pS2, cathepsin D, cyclin D1, c-Myc, and the progesterone receptor, are positively regulated by estrogen treatment (21). Transcriptional repression by ER has not been as well studied. However, using Serial Analysis of Gene Expression (SAGE) and DNA microarrays, many more estrogen-responsive genes, induced and repressed by the hormone, have been identified and characterized (22-29). Much of the work on gene expression has been focused on the role of ERα, but little is known about genes specifically targeted by ERβ or by α/β heterodimers. Recent microarray experiments using knock-out animals indicate that target tissues in wild-type controls exhibited an overall reduced transcriptional response to hormone treatment as compared to ERβ knockouts (30). Expression studies of osteosarcoma cells stably transfected with each receptor subtype suggest that ERα and ERβ share some common target genes, although each also appear to have distinct sets of downstream targets (31). Despite these efforts the exact transcriptional effects of ERα and ERβ remain obscure.
[0010]ERβ has also been shown to antagonize the growth of ERα-positive hormone-dependent cultured breast tumor cells. ERβ function is currently inferred from its transcript (real-time PCR) and protein levels (ligand-binding and immunohistochemical assays). However the association of ERβ expression with breast cancer prognosis has been difficult to establish using transcript and protein levels as measures of ERβ activity, as increased gene expression does not necessarily lead to increased gene function and subsequent alteration in tumor behavior in vivo.
SUMMARY OF THE INVENTION
[0011]The present inventors have identified cell cycle and DNA replication genes which are suppressed by ERβ overexpression in ERα-positive breast tumor cells. Expression profiles of these genes and ERβ in ERα-positive tumors from individuals who received adjuvant tamoxifen therapy revealed statistically significant inverse correlation of four genes, CDC2, CDC6, CKS2 and DNA2L, with ERβ transcript levels. Moreover, the expression profiles of this expression, cassette (five genes, including ERβ are associated with tumor grade, 10-year disease recurrence, and patient survival. Therefore the in vivo behavior of these genes appears to be associated with ERβ function and is predictive of disease outcome in breast cancer individuals treated with adjuvant endocrine therapy.
[0012]High-density DNA microarrays were employed to generate genome-wide expression profiles of genes responsive to ERβ expression and estrogen treatment in ERα-positive breast tumor cells. The expression profiles provided insights into the interactions between ERα and ERβ in regulating hormone response, and the potential roles of putative ERβ-modulated genes in the biology of primary breast tumors.
[0013]Accordingly, the present invention provides means to correlate ERβ expression and function with tumor behavior, thereby enabling better prognosis of clinical outcomes and conseqnent reduction in morbidity and mortality from breast cancers. More specifically, the present invention provides a gene expression cassette that is associated with both ERβ expression level and its transcriptional effects on downstream-cell cycle and DNA replication genes. Also provided are pharmaceutical compositions for treating breast cancers, and methods, kits and reagents for predicting outcomes and treating breast tumors.
[0014]In a first aspect of the present invention, there is provided a method of assessing ER-β function, the method comprising: [0015](i) determining the level of at least one marker selected from the group consisting of CDC2; CDC6, DNA2L and CKS2; and [0016](ii) using the level of the at least one said marker as an indication of ER-β function.
[0017]In one embodiment, the method defined in the first aspect of the present invention may be used for: (i) assessing treatment efficacy or cancer progression or regression; (ii) identifying a compound useful for the treatment of cancer; or (iii) assessing the carcinogenic potential of a test compound.
[0018]In another embodiment, the method may comprise determining the level of the marker(s) in a patient sample. In a preferred embodiment, the sample may be ER-α positive.
[0019]In another embodiment, the sample may be derived from a patient suffering from cancer. The cancer may, for example, be breast cancer, prostate cancer or colon cancer. The sample may be obtained at one or more time points. Expression levels of the marker(s) may optionally be compared with a control sample. The control sample may be a sample derived from a person not having cancer (e.g. breast cancer, prostate cancer or colon cancer). One or more control samples may be employed.
[0020]In a second aspect of the present invention; there is provided a method for cancer prognosis, the method comprising: [0021](i) determining the level of at least one marker selected from the group consisting of CDC2, CDC6, DNA2L and CKS2 in an ER-α positive sample of a patient; and [0022](ii) using the level of the at least one said marker in making a disease prognosis for the patient.
[0023]In a third aspect of the present invention, there is provided a method of selecting a treatment for a cancer patient, the method comprising: [0024](i) determining the level of at least one marker selected from the group consisting of CDC2, CDC6, DNA2L and CKS2 in a ER-α positive sample of the patient; and [0025](ii) using the level of the at least one said marker in selecting an appropriate treatment for the patient.
[0026]In a fourth aspect of the present invention, there is provided a method of characterizing a cancerous cell or a cell suspected of being cancerous, the method comprising: [0027](i) determining the level of at least one marker selected from the group consisting of CDC2, CDC6, DNA2L and CKS2; and [0028](ii) using the level of the at least one marker in the characterization of the cell(s); wherein the cell(s) are ER-α positive.
[0029]In some embodiments of the methods described above, the level of at least two or three markers selected from the group consisting of CDC2, CDC6, DNA2L and CKS2 may be determined. In one embodiment, the level of CDC2, CDC6, DNA2L and CKS2 may be determined.
[0030]In some embodiments, the cancer may be selected from the group consisting of breast cancer, prostate cancer and colon cancer.
[0031]In another embodiment, the level of a further marker may be determined, i.e. a marker other than a CDC2, CDC6; DNA2L and CKS2. The further marker may optionally be selected from Table 4 or may be ER-β.
[0032]In another embodiment, the cancer may be treated with adjuvant endocrine therapy.
[0033]In a fifth aspect of the present invention, there is provided an array for assessing ER-β function, comprising probes for determining the level of at least one, two or three markers selected from the group consisting of CDC2, CDC6, DNA2L and CKS2.
[0034]In one embodiment, the array may comprise probes for determining the level of CDC2, CDC6, DNA2L and CKS2. In one embodiment, the array may comprise probes which are bound to nucleic acids or proteins derived from a sample (e.g. a cell) which is suspected of being cancerous or which is known to be cancerous. The derived nucleic acids or proteins are not necessarily physically, derived from the sample but may be obtained in any manner including, for example, DNA replication (e.g. by PCR). Hence a broad interpretation of the phrase "derived from" is intended. Thus, the array in "use", i.e. an array that is bound to nucleic acids or proteins derived from a sample is encompassed.
[0035]It will be appreciated that one embodiment of the invention provides for the use of the array in the methods of the invention in determining the level of the marker(s).
[0036]In a sixth aspect of the present invention, there is provided a kit for assessing ER-β function, comprising a reagent for determining the level of at least one, two or three markers selected from the group consisting of CDC2, CDC6, DNA2L and CKS2. In some embodiments, reagents for determining the level of CDC2, CDC6, DNA2L and CKS2 may be provided.
[0037]Details of the aspects and embodiments of the invention described herein are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
DEFINITIONS
[0038]The following words and phrases (and where appropriate grammatical variants thereof) may be defined as set forth below.
[0039]As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.
[0040]An individual is not limited to a human being but may comprise other organisms including but not limited to vertebrates and invertebrates or one or more cells derived from the same. Examples of individuals include any animal, including rodents (e.g. mice, rats, guinea pigs, hamster), rabbits, cats, dogs, pigs, goats, sheep, monkeys (or other primates), horses, cows, etc. The individual may be male or female. The female may be pre or post menopausal. The individual may be a cancer patient. The individual may be a clinical patient, a clinical trial volunteer, an experimental animal etc. The individual may be in need of treatment, for example by surgery, therapy, diagnosis or prognosis etc. The individual may, for example, be suspected of suffering from cancer, or suspected of being predisposed to cancer, or may have previously suffered from cancer or may currently be suffering from cancer. In one embodiment the cancer is a primary cancer. In one embodiment the cancer is selected from the group consisting of breast cancer, prostate cancer and colon cancer. In one embodiment the cancer is primary breast cancer. In one embodiment the individual has received adjuvant endocrine therapy (and may optionally still be receiving adjuvant endocrine therapy) or has been identified as being a suitable patient for adjuvant endocrine therapy (e.g. on the basis of ER-α status). The terms individual and patient are used herein interchangeably.
[0041]As used herein, the term "comprising" means "including." Variations of the word "comprising", such as "comprise" and "comprises," have correspondingly varied meanings. Thus, for example, a composition "comprising" X may consist exclusively of X or may include one or more additional components.
[0042]The term "antibody" means an immunoglobulin molecule able to bind to a specific epitope on an antigen. Antibodies can be comprised of a polyclonal mixture, or may be monoclonal in nature. Further, antibodies can be entire immunoglobulins derived from natural sources, or from recombinant sources. The antibodies of the present invention may exist in a variety of forms, including for example as a whole antibody, or as an antibody fragment, or other immunologically active fragment thereof, such as complementarity determining regions. Similarly, the antibody may exist as an antibody fragment having functional antigen-binding domains, that is, heavy and light chain variable domains. Also, the antibody fragment may exist in a form selected from the group consisting of, but not limited to: Fv, Fab, F(ab)2; scFv (single chain Fv), dAb (single domain antibody), bi-specific antibodies, diabodies and triabodies.
[0043]As used herein, an "array" includes an intentionally created collection of molecules (e.g. probes) which can be prepared either synthetically or biosynthetically. The molecules in the array can be identical or different from each other. The array can assume a variety of formats, e.g., libraries of soluble molecules; libraries of compounds tethered to resin beads, silica chips, or other solid supports. The term "array" includes, inter alia, those libraries of nucleic acids which can be prepared by spotting nucleic acids of essentially any length (e.g., from 1 to about 1000 nucleotide monomers in length) onto a substrate. As used herein, the term array and microarray may be used interchangeably.
[0044]Throughout this disclosure, various, aspects of this invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
[0045]The term "complementary" refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single stranded RNA or DNA molecules are said to be complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%; and more preferably from about 98 to 100% of the nucleotides of the other strand. Alternatively, complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65% complementarity over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, and more preferably at least about 90% complementarity.
[0046]As used herein, the term "determining" is intended to include measuring the expression level (e.g. the amount or concentration of) a marker (e.g. nucleic acid or protein) of the invention. The term thus refers to the use of the materials, compositions and methods of the present invention for qualitative, and quantitative determination. By determining the level of a particular marker, we include qualitative determination (e.g. comparing the level of the marker in a sample with the level of the marker of a control sample or with the level of the marker from a sample obtained from the same patient but at a different time point) or quantitative determination (e.g. measuring the amount or concentration) of the level of a nucleic acid or protein which is encoded by or which corresponds to the particular marker (marker nucleic acids and marker proteins, respectively). For example, detecting a change in expression levels may include quantifying a change of any value between 10% and 90%, or of any value between 30% and 60%, or over 100%, of a marker of the invention when compared to a control. In other embodiments, detecting an increase in gene expression levels may include quantifying a change of any value between 1 to 5 fold or more of any of the markers of the invention when compared to a control.
[0047]As used herein "Disease-specific survival" or DSS refers to a survival assessment where the end point being examined is death because of a specific disease, for example, breast cancer. DSS refers to survival from a particular disease for a defined period of time and is usually reported as time since diagnosis or treatment. Death from some other cause is not considered when calculating DSS.
[0048]As used herein, "Disease-free survival" or DFS refers to a survival assessment where the end points are either tumor recurrence (e.g., the cancer comes back as a consequence of distant metastasis, to other sites in the body) or death (for example, because of breast cancer without evidence of distant metastasis). The term "disease-free-survival" as used herein is defined as a time between diagnosis or surgery to treat a cancer patient and reoccurrence. For example, a disease-free survival is "low" if the cancer patient has a first reoccurrence within five years after tumor resection, and more specifically, if the cancer patient has less than about 55% disease-free survival over 5 years. For example, a high disease-free survival refers to at least about 55% disease-free survival over 5 years.
[0049]The term "endocrine therapy" as used herein is defined as a treatment of or pertaining to any of the ducts or endocrine glands characterized by secreting internally end into the bloodstream from the cells of the gland. The treatment may remove the gland, block hormone synthesis, or prevent the hormone from binding to its receptor. Non-limiting examples of endocrine therapies that are contemplated by the present invention include tamoxifen, raloxifene, or other SERMs (selective estrogen-receptor modulators). Tamoxifen has been the most commonly prescribed drug to treat breast cancer since its approval by the U.S. Food and Drug Administration (FDA) in the 1970s. Tamoxifen is an anti-estrogen and works by competing with the hormone estrogen to bind to estrogen receptors in breast cancer cells. Tamoxifen has been shown to reduce the risk of recurrence of an original cancer and the risk of developing new cancers by working against the effects of estrogen on breast cancer cells. A pharmaceutical composition comprising tamoxifen is generally administered as an oral composition such as a pill or capsule. Tamoxifen belongs to a class of agents known as selective estrogen receptor modulators. These agents display estrogen antagonist activity on some genes and agonist activity on others.
[0050]As used herein, a "fragment," or "segment," refers to a portion of a larger polynucleotide that is capable of being differentially expressed or detected in an assay or screening method according to the invention. A polynucleotide, for example, can be broken up, or fragmented into, a plurality of segments. Various methods of fragmenting nucleic acids are well known in the art. These methods may be, for example, either chemical or physical in nature. Chemical fragmentation may include partial degradation with a nuclease e.g. DNase; partial depurination with acid; the use of restriction enzymes; intron-encoded endonucleases; DNA-based cleavage methods, such as triplex and hybrid formation methods; that rely on the specific hybridization of a nucleic acid segment to localize a cleavage agent to a specific location in the nucleic-acid molecule; or other enzymes or compounds which cleave DNA at known or unknown locations. Physical fragmentation methods may involve subjecting the DNA to a high shear rate. High shear rates may be produced, for example, by moving DNA through a chamber or channel with pits or spikes, or forcing the DNA sample through a restricted size flow passage, e.g., an aperture having a cross sectional dimension in the micron or submicron scale. Other physical methods include sonication and nebulization. Combinations of physical and chemical fragmentation methods may likewise be employed such as fragmentation by heat and ion-mediated hydrolysis. See for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual," 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) ("Sambrook et al.") which is incorporated herein by reference for all purposes. These methods can be optimized to digest a nucleic acid into fragments of a selected size range. Useful size ranges may be from 10, 15, 25, 50, 100, 200, 400, 700 or 1000 to 500, 800, 1500, 2000, 4000 or 10,000 base pairs. However, larger size ranges such as 4000, 10,000 or 20,000 to 10,000, 20,000 or 500,000 base pairs may also be useful.
[0051]As used-herein, the term "hybridization" refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. The term "hybridization" may also refer to triple-stranded hybridization. The resulting (usually) double-stranded polynucleotide is a "hybrid."The proportion of the population of polynucleotides that forms stable hybrids is referred to herein as the "degree of hybridization."
[0052]Hybridization conditions will typically include salt concentrations of less than about 1M, more usually less than about 500 mM and less than about 200 mM. Hybridization temperatures can be as low as 5° C., but are typically greater than 22° C., more typically greater than about 30° C., and preferably in excess of about 37° C. Hybridizations are usually performed under stringent conditions, i.e. conditions under which a probe will hybridize to its target subsequence. Stringent conditions are sequence-dependent and are different under different circumstances. Longer fragments may require higher hybridization temperatures for specific hybridization. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, combination of parameters is more important than the absolute measure of any one alone. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid composition) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Typically, stringent conditions include salt concentration of at least 0.01 M to no more than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and a temperature of at least 25° C. For example, conditions of 5×SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30° C. are suitable for allele-specific probe hybridizations. For stringent conditions, see for example, Sambrook, Fritsche and Maniatis. "Molecular Cloning A laboratory Manual" 2nd Ed. Cold Spring Harbor press (1989) and Anderson "Nucleic Acid Hybridization" 1st Ed., BIOS Scientific Publishers Limited (1999), which are hereby incorporated by reference in their entireties for all purposes above.
[0053]The term "probe" refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example, a nucleotide transcript or protein. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules. In some embodiments, a probe can be surface immobilized. Where nucleic acids (such as oligonucleotides) are used they may be capable of binding in a base-specific manner to another strand of nucleic acid. Hybridization may occur between complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch. Such probes include peptide nucleic acids, as described in Nielsen et al., Science 254:1497-1500 (1991); Nielsen Curr. Opin. Biotechnol., 10:71-75 (1999) and other nucleic acid analogs and nucleic acid mimetics.
[0054]As used herein, "mRNA" includes, but is not limited to, pre-mRNA transcript(s), transcript processing intermediates, mature mRNA(s) ready for translation and transcripts of the gene or genes, or nucleic acids derived from the mRNA transcript(s). Transcript processing may include splicing, editing and degradation. As used herein, a nucleic acid derived from an mRNA transcript refers to a nucleic acid for whose synthesis the mRNA transcript or a subsequence thereof has ultimately served as a template. Thus, a cDNA reverse transcribed from an mRNA, a cRNA from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, etc., are all derived from the mRNA transcript and detection of such derived products is indicative of the presence and/or abundance of the original transcript in a sample. Thus, mRNA derived samples include, but are not limited to, mRNA transcripts of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA transcribed from the cDNA, DNA amplified from the genes, RNA transcribed from amplified DNA, and the like.
[0055]As used herein, the term "nucleic acid", and equivalent terms such as polynucleotide, refers to a polymeric form of nucleotides of any length, such as ribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs), that comprise purine and pyrimidine bases; or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The nucleic acid may be double stranded or single stranded. References to single stranded nucleic acids include references to the sense or antisense strands. The backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. The terms nucleoside, nucleotide, deoxynucleoside and deoxynucleotide generally include complements, fragments and variants of the nucleoside, nucleotide, deoxynucleoside and deoxynucleotide, or analogs thereof.
[0056]A "variant" is a nucleic acid molecule that is a recognized variation of a nucleic acid molecule or expression product thereof. Splice variants may be determined for example by using computer programs, e.g., BLAST. Allelic variants have in general a high percent identity to the nucleic acid molecule of interest. "Single nucleotide polymorophism" (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid.
[0057]Analogs are those molecules having some structural features in common with a naturally occurring nucleoside or nucleotide such that when incorporated into a nucleic acid or oligonucleotide sequence, they allow hybridization with a naturally occurring nucleic acid sequence in solution. Typically, these analogs are derived from naturally occurring nucleosides and nucleotides by replacing and/or modifying the base, the ribose or the phosphodiester moiety. Analogs in general retain the biological activities of the naturally-occurring molecules but may confer advantages such as longer lifespan or enhanced activity. The changes can be tailor made to stabilize or destabilize hybrid formation or enhance the specificity of hybridization with a complementary nucleic acid sequence as desired.
[0058]The term "labeled", with regard to, for example, a probe, is intended to encompass direct labeling of the probe by coupling (i.e., physically linking) a detectable substance to the probe, as well as indirect labeling of the probe by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
[0059]An "oligonucleotide" as used herein is a single stranded molecule which may be used in hybridization or amplification technologies. In general, an oligonucleotide may be any integer from about 15 to about 100 nucleotides in length, but may also be of greater length.
[0060]As used herein, "solid support", "support", and "substrate" are used interchangeably and include a reference to a material or group of materials which may have a rigid or semi-rigid surface or surfaces. In many embodiments, at least one surface of the solid support will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different compounds with, for example, wells, raised regions, pins, etched trenches, or the like. According to other embodiments, the solid support(s) may take the form of beads, resins, gels, microspheres, or other, geometric configurations. Examples of supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. See also U.S. Pat. No. 5,744,305 for exemplary substrates.
[0061]As used herein, the term "sample" includes tissues, cells, body fluids and isolates thereof etc., isolated from a subject, as well as tissues, cells and fluids etc. present within a subject (i.e. the sample is in vivo). Examples of samples include: blood fluids, lymph and cystic fluids, as well as nipple aspirates, sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, archival samples, blood, plasma, serum, sputum, stool, tears, mucus, hair, skin, ascitic fluid, cystic fluid, urine, nipple exudate, explants and primary and/or transformed cell cultures derived from patient tissues etc. In one embodiment the sample may be a "breast-associated" body fluid, which is a fluid which, when in the body of a patient, contacts or passes through breast cells, or into which cells, nucleic acids or proteins shed from breast cells are capable of passing. Examples of breast-associated body fluids include blood fluids, lymph, cystic fluid, and nipple aspirates.
[0062]As used herein the term "treatment", refers to any and all methods which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. The term "treatment" includes, inter alia: (i) the prevention or inhibition of cancer or cancer recurrence, (ii) the reduction or elimination of symptoms or cancer cells, and (iii) the substantial or complete elimination of the cancer in question. Treatment may be effected prophylactically or therapeutically. Treatment may entail treatment with a single agent or with a combination (more than two) of agents. An "agent" is used herein broadly to refer to, for example, a compound or other means for treatment e.g. radiation treatment or surgery.
[0063]Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0064]The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings will be provided by the Patent Office upon request and payment of the necessary fee.
[0065]The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the disclosed principles of the invention.
[0066]FIG. 1. Similar expression profiles of responsive genes under all induction and treatment conditions. (A) 1847 genes up-regulated by ERβ expression (α+β; left panel), E2 treatment (α, +E2; center panel), or both in combination (α+β, +E2; right panel), as determined by fold-change ratios over untreated-uninduced controls (see Materials and Methods), were visualized by hierarchical clustering and the Eisen TreeView software. The columns represent time points arranged in chronological order, and each row represents the expression profile of a particular gene. By convention, up-regulated genes are indicated by red signals and down-regulated genes are indicated by green. The magnitude of change is proportional to the brightness of the signal. (B) Expression profiles of 1662 down-regulated responsive genes. Expression profiles of cell cycle and DNA replication genes differentially regulated by ERβ expression are highlighted by magenta colored boxes.
[0067]FIG. 2. Models of ligand-dependent interaction between ERα and ERβ. Differential regulation of gene expression by ERβ, in the presence of hormone ligand (E2+ERβ), as compared to ERα alone (E2). Responsive genes are divided into synergistic (A), attenuation (B), and antagonistic (C) models of interaction between receptor subtypes. Synergistic genes were defined by an increase in the magnitude of change in the same direction when ERβ was expressed and cells treated with E2 as compared to E2 treatment alone. The attenuation model includes genes whose expression levels were diminished by ERβ expression. In the antagonistic model, genes responded in opposite directions in ERβ expressing cells treated with E2 as compared to the hormone treated controls.
[0068]FIG. 3. Validation of estrogen-responsive regulation of cell cycle and DNA replication genes suppressed by ERβ over-expression by real-time quantitative PCR. CDC2, CDC6, DNA2L and CKS2 were selected for further validation based on their significant correlation with ERβ transcript levels and β actin expression was assessed as a negative control. Transcript levels in T-47 Dbeta cells were measured at 30 hours following estrogen treatment (+E2) or mock treatment and under induction conditions [-TET (+ERβ)] and non-induction (+TET) conditions. Relative fold-changes were calculated using the non-induced (+TET) samples as the reference.
[0069]FIG. 4. Clustering of 69 tumor samples by ERβ/ESR2, CDC2, CDC6, DNA2L and CKS2 expression profiles is associated with clinical parameters and disease outcome. (A) Hierarchical clustering of tumor samples into two groups of high (black dendrogram; Cluster 1) and low (red dendrogram; Cluster 2) expression clusters. Clinical parameters, including relapse and death from breast cancer within 10 years of surgery, and lymph node positive status are indicated with a solid bar beneath each tumor sample. Tumor grade is indicated by colored bars with green, blue, and red bars denoting grades 1, 2, and 3 respectively. Gray bars denote missing data. Significant distribution of clinical parameters between the two clusters was determined by Chi-square tests and P-values derived from the calculated distributions. Kaplan-Meier plots of (B) DFS and (C) DSS curves for Cluster 1 (black) and Cluster 2 (red) patients are shown with the associated P-values from likelihood-ratio analysis.
[0070]FIG. 5. Clustering of 45 previously published ERα-positive tumor samples (36) using the available genes from the ERβ expression cassette expression profile is associated with disease outcome. (A) Hierarchical clustering of tumor samples into two groups of high (black dendrogram) and low (red dendrogram) expression clusters. Kaplan-Meier plots of (B) DFS and (C) DSS curves for high (black) and low (red) expression patients are shown with the associated P-values. ERβ levels were not assessed due to the absence of ERβ probes on the arrays used by Sotiriou et al. (36).
DETAILED DESCRIPTION OF THE INVENTION
[0071]When a patent, application, or other reference is cited or repeated below, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited.
[0072]The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the example herein below. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al: (2002) Biochemistry, 5th Ed., W.H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.
[0073]The present inventors have discovered a transcriptionally-regulated mechanism for the growth inhibitory effects of ERβ in ERα-positive breast tumor cells. The functional characterization of the downstream targets of ER-β transcriptional regulation provides strong evidence for a functional and beneficial impact of ER-β on the in vivo behavior of primary breast cancers.
[0074]In the present invention, the level of at least one, two or three markers selected from the group consisting of CDC2, CDC6, DNA2L and CKS2 is determined.
[0075]In one embodiment the level of at least CDC2 and CDC6; CDC2 and DNA2L; CDC2 and CKS2; CDC6 and DNA2L; CDC6 and CKS2; or DNA2L and CKS2 is determined. In one embodiment the level of at least CDC2, CDC6, and DNA2L; CDC2, CDC6, and CKS2; CDC2, DNA2L and CKS2; or CDC6, DNA2L and CKS2 is determined.
[0076]In one embodiment, the level of one, two, three or all four of CDC2, CDC6, DNA2L and CKS2 is determined.
[0077]For the avoidance of doubt, a reference to "a marker of the invention" or "one or more of the markers of the invention" etc. includes a reference to one, two, three or all four of CDC2, CDC6, DNA2L and CKS2 and optionally also ER-β. It also includes a reference to a "marker protein" and a "marker nucleic acid."
[0078]The markers of the invention may be identified by their Accession numbers. Accession numbers, as used herein, may refer to Accession numbers from multiple databases, including GenBank, the European Molecular Biology Laboratory (EMBL), the DNA Database of Japan (DDBJ), or the Genome Sequence Database (GSDB), for nucleotide sequences, and including the Protein Information Resource. (PIR), SWISSPROT, Protein Research Foundation (PRF), and Protein Data Bank (PDB) (sequences from solved structures), as well as from translations from annotated coding regions from nucleotide sequences in GenBank, EMBL, DDBJ, or RefSeq, for polypeptide sequences. Accession numbers as used herein, may also refer to Accession numbers from databases such as UniGene, OMIM, LocusLink, or HomologoGene.
[0079]The sequences of the accession numbers are hereby expressly incorporated by reference. GenBank is available, see, e.g., Benson, et al. (1998) Nuc. Acids Res. 26:1-7; and http://www.ncbi.nhn.nih.gov/. Sequences are also available in other databases, 25 e.g., European Molecular Biology Laboratory (EMBL) and DNA Database of Japan (DDBJ).
[0080]As indicated in Table 1 below, reference to CDC2, CDC6, DNA2L, CKS2, and ER-β includes allelic variants of CDC2, CDC6, DNA2L, CKS2 and ER-β, complements of CDC2, CDC6, DNA2L, CKS2 and ER-β, as well as to the non-human counterparts (i.e. homologs) of CDC2, CDC6, DNA2L, CKS2 and ER-β. References to other markers are intended to be construed in a likewise manner.
TABLE-US-00001 TABLE 1 Human Human Gene Variant 1 Variant 2 Chimpanzee Mouse Rat Fruit Fly Worm ER-β NM_001437 NM_207707 NM_012754 CDC2 NM_001786 NM_033379 XM_507809 NM_007659 NM_019296 NM_057449 NM_171224 (SEQ ID NO. 1) (SEQ ID NO. 3) NP_001777 NP_203698 (SEQ ID NO. 2) (SEQ ID NO. 4) CDC6 NM_001254 XM_523625 NM_011799 XM_340896 NM_058942 (SEQ ID NO. 5) NP_001245 (SEQ ID NO. 6) CKS2 NM_001827 XM_520119 NM_025415 NM_141560 (SEQ ID NO. 7) NP_001818 (SEQ ID NO. 8) DNA2L XM_166103 NM_177372 XM_241671 NM_132369 NM_064115 (SEQ ID NO. 9) XP_166103 (SEQ ID NO. 10)
[0081]The sequences of SEQ ID Nos. 1-10 are shown below:
TABLE-US-00002 SEQ ID NO. 1 >gi|16306490|ref|NM_001786.2| Homo sapiens cell division cycle 2, G1 to S and G2 to M (CDC2), transcript variant 1, mRNA GGGGGGGGGGGGCACTTGGCTTCAAAGCTGGCTCTTGGAAATTGAGCGGA GAGCGACGCGGTTGTTGTAGCTGCCGCTGCGGCCGCCGCGGAATAATAAG CCGGGATCTACCATACCCATTGACTAACTATGGAAGATTATACCAAAATA GAGAAAATTGGAGAAGGTACCTATGGAGTTGTGTATAAGGGTAGACACAA AACTACAGGTCAAGTGGTAGCCATGAAAAAAATCAGACTAGAAAGTGAAG AGGAAGGGGTTCCTAGTACTGCAATTCGGGAAATTTCTCTATTAAAGGAA CTTCGTCATCCAAATATAGTCAGTCTTCAGGATGTGCTTATGCAGGATTC CAGGTTATATCTCATCTTTGAGTTTCTTTCCATGGATCTGAAGAAATACT TGGATTCTATCCCTCCTGGTCAGTACATGGATTCTTCACTTGTTAAGAGT TATTTATACCAAATCCTACAGGGGATTGTGTTTTGTCACTCTAGAAGAGT TCTTCACAGAGACTTAAAACCTCAAAATCTCTTGATTGATGACAAAGGAA CAATTAAACTGGCTGATTTTGGCCTTGCCAGAGCTTTTGGAATACCTATC AGAGTATATACACATGAGGTAGTAACACTCTGGTACAGATCTCCAGAAGT ATTGCTGGGGTCAGCTCGTTACTCAACTCCAGTTGACATTTGGAGTATAG GCACCATATTTGCTGAACTAGCAACTAAGAAACCACTTTTCCATGGGGAT TCAGAAATTGATCAACTCTTCAGGATTTTCAGAGCTTTGGGCACTCCCAA TAATGAAGTGTGGCCAGAAGTGGAATCTTTACAGGACTATAAGAATACAT TTCCCAAATGGAAACCAGGAAGCCTAGCATCCCATGTCAAAAACTTGGAT GAAAATGGCTTGGATTTGCTCTCGAAAATGTTAATCTATGATCCAGCCAA ACGAATTTCTGGCAAAATGGCACTGAATCATCCATATTTTAATGATTTGG ACAATCAGATTAAGAAGATGTAGCTTTCTGACAAAAAGTTTCCATATGTT ATGTCAACAGATAGTTGTGTTTTTATTGTTAACTCTTGTCTATTTTTGTC TTATATATATTTCTTTGTTATCAAACTTCAGCTGTACTTCGTCTTCTAAT TTCAAAAATATAACTTAAAAATGTAAATATTCTATATGAATTTAAATATA ATTCTGTAAATGTGAAAAAAAAAAAAAAAAAAAAA SEQ ID NO. 2 >gi|4502709|ref|NP_001777.1| cell division cycle 2 protein isoform 1 [Homo sapiens] MEDYTKIEKIGEGTYGVVYKGRHKTTGQVVAMKKIRLESEEEGVPSTAIR EISLLKELRHPNIVSLQDVLMQDSRLYLIFEFLSMDLKKYLDSIPPGQYM DSSLVKSYLYQILQGIVFCHSRRVLHRDLKPQNLLIDDKGTIKLADFGLA RAFGIPIRVYTHEVVTLWYRSPEVLLGSARYSTPVDIWSIGTIFAELATK KPLFHGDSEIDQLFRIFRALGTPNNEVWPEVESLQDYKNTFPKWKPGSLA SHVKNLDENGLDLLSKMLIYDPAKRISGKMALNHPYFNDLDNQIKKM SEQ ID NO. 3 >gi|27886643|ref|NM_033379.2| Homo sapiens cell division cycle 2, G1 to S and G2 to M (CDC2), transcript variant 2, mRNA CCATTGACTAACTATGGAAGATTATACCAAAATAGAGAAAATTGGAGAAG GTACCTATGGAGTTGTGTATAAGGGTAGACACAAAACTACAGGTCAAGTG GTAGCCATGAAAAAAATCAGACTAGAAAGTGAAGAGGAAGGGGTTCCTAG TACTGCAATTCGGGAAATTTCTCTATTAAAGGAACTTCGTCATCCAAATA TAGTCAGTCTTCAGGATGTGCTTATGCAGGATTCCAGGTTATATCTCATC TTTGAGTTTCTTTCCATGGATCTGAAGAAATACTTGGATTCTATCCCTCC TGGTCAGTACATGGATTCTTCACTTGTTAAGGTAGTAACACTCTGGTACA GATCTCCAGAAGTATTGCTGGGGTCAGCTCGTTACTCAACTCCAGTTGAC ATTTGGAGTATAGGCACCATATTTGCTGAACTAGCAACTAAGAAACCACT TTTCCATGGGGATTCAGAAATTGATCAACTCTTCAGGATTTTCAGAGCTT TGGGCACTCCCAATAATGAAGTGTGGCCAGAAGTGGAATCTTTACAGGAC TATAAGAATACATTTCCCAAATGGAAACCAGGAAGCCTAGCATCCCATGT CAAAAACTTGGATGAAAATGGCTTGGATTTGCTCTCGAAAATGTTAATCT ATGATCCAGCCAAACGAATTTCTGGCAAAATGGCACTGAATCATCCATAT TTTAATGATTTGGACAATCAGATTAAGAAGATGTAGCTTTCTGACAAAAA GTTTCCATATGTTATGTCAACAGATAGTTGTGTTTTTATTGTTAACTCTT GTCTATTTTTGTCTTATATATATTTCTTTGTTATCAAACTTCAGCTGTAC TTCGTCTTCTAATTTCAAAAATATAACTTAAAAATGTAAATATTCTATAT GAATTTAAATATAATTCTGTAAATGTGAAAAAAAAAAAAAAAAAAAAA SEQ ID NO. 4 >gi|16306492|ref|NP_203698.1| cell division cycle 2 protein isoform 2 [Homo sapiens] MEDYTKIEKIGEGTYGVVYKGRHKTTGQVVAMKKIRLESEEEGVPSTAIR EISLLKELRHPNIVSLQDVLMQDSRLYLIFEFLSMDLKKYLDSIPPGQYM DSSLVKVVTLWYRSPEVLLGSARYSTPVDIWSIGTIFAELATKKPLFHGD SEIDQLFRIFRALGTPNNEVWPEVESLQDYKNTFPKWKPGSLASHVKNLD ENGLDLLSKMLIYDPAKRISGKMALNHPYFNDLDNQIKKM SEQ ID NO. 5 >gi|51944959|ref|NM_001254.3| Homo sapiens CDC6 cell division cycle 6 homolog (S. cerevisiae) (CDC6), mRNA GAGCGCGGCTGGAGTTTGCTGCTGCCGCTGTGCAGTTTGTTCAGGGGCTT GTGGTGGTGAGTCCGAGAGGCTGCGTGTGAGAGACGTGAGAAGGATCCTG CACTGAGGAGGTGGAAAGAAGAGGATTGCTCGAGGAGGCCTGGGGTCTGT GAGGCAGCGGAGCTGGGTGAAGGCTGCGGGTTCCGGCGAGGCCTGAGCTG TGCTGTCGTCATGCCTCAAACCCGATCCCAGGCACAGGCTACAATCAGTT TTCCAAAAAGGAAGCTGTCTCGGGCATTGAACAAAGCTAAAAACTCCAGT GATGCCAAACTAGAACCAACAAATGTCCAAACCGTAACCTGTTCTCCTCG TGTAAAAGCCCTGCCTCTCAGCCCCAGGAAACGTCTGGGCGATGACAACC TATGCAACACTCCCCATTTACCTCCTTGTTCTCCACCAAAGCAAGGCAAG AAAGAGAATGGTCCCCCTCACTCACATACACTTAAGGGACGAAGATTGGT ATTTGACAATCAGCTGACAATTAAGTCTCCTAGCAAAAGAGAACTAGCCA AAGTTCACCAAAACAAAATACTTTCTTCAGTTAGAAAAAGTCAAGAGATC ACAACAAATTCTGAGCAGAGATGTCCACTGAAGAAAGAATCTGCATGTGT GAGACTATTCAAGCAAGAAGGCACTTGCTACCAGCAAGCAAAGCTGGTCC TGAACACAGCTGTCCCAGATCGGCTGCCTGCCAGGGAAAGGGAGATGGAT GTCATCAGGAATTTCTTGAGGGAACACATCTGTGGGAAAAAAGCTGGAAG CCTTTACCTTTCTGGTGCTCCTGGAACTGGAAAAACTGCCTGCTTAAGCC GGATTCTGCAAGACCTCAAGAAGGAACTGAAAGGCTTTAAAACTATCATG CTGAATTGCATGTCCTTGAGGACTGCCCAGGCTGTATTCCCAGCTATTGC TCAGGAGATTTGTCAGGAAGAGGTATCCAGGCCAGCTGGGAAGGACATGA TGAGGAAATTGGAAAAACATATGACTGCAGAGAAGGGCCCCATGATTGTG TTGGTATTGGACGAGATGGATCAACTGGACAGCAAAGGCCAGGATGTATT GTACACGCTATTTGAATGGCCATGGCTAAGCAATTCTCACTTGGTGCTGA TTGGTATTGCTAATACCCTGGATCTCACAGATAGAATTCTACCTAGGCTT CAAGCTAGAGAAAAATGTAAGCCACAGCTGTTGAACTTCCCACCTTATAC CAGAAATCAGATAGTCACTATTTTGCAAGATCGACTTAATCAGGTATCTA GAGATCAGGTTCTGGACAATGCTGCAGTTCAATTCTGTGCCCGCAAAGTC TCTGCTGTTTCAGGAGATGTTCGCAAAGCACTGGATGTTTGCAGGAGAGC TATTGAAATTGTAGAGTCAGATGTCAAAAGCCAGACTATTCTCAAACCAC TGTCTGAATGTAAATCACCTTCTGAGCCTCTGATTCCCAAGAGGGTTGGT CTTATTCACATATCCCAAGTCATCTCAGAAGTTGATGGTAACAGGATGAC CTTGAGCCAAGAAGGAGCACAAGATTCCTTCCCTCTTCAGCAGAAGATCT TGGTTTGCTCTTTGATGCTCTTGATCAGGCAGTTGAAAATCAAAGAGGTC ACTCTGGGGAAGTTATATGAAGCCTACAGTAAAGTCTGTCGCAAACAGCA GGTGGCGGCTGTGGACCAGTCAGAGTGTTTGTCACTTTCAGGGCTCTTGG AAGCCAGGGGCATTTTAGGATTAAAGAGAAACAAGGAAACCCGTTTGACA AAGGTGTTTTTCAAGATTGAAGAGAAAGAAATAGAACATGCTCTGAAAGA TAAAGCTTTAATTGGAAATATCTTAGCTACTGGATTGCCTTAAATTCTTC TCTTACACCCCACCCGAAAGTATTCAGCTGGCATTTAGAGAGCTACAGTC TTCATTTTAGTGCTTTACACATTCGGGCCTGAAAACAAATATGACCTTTT TTACTTGAAGCCAATGAATTTTAATCTATAGAITCTTTAATATTAGCACA GAATAATATCTTTGGGTCTTACTATTTTTACCCATAAAAGTGACCAGGTA GACCCTTTTTAATTACATTCACTACTTCTACCACTTGTGTATCTCTAGCC AATGTGCTTGCAAGTGTACAGATCTGTGTAGAGGAATGTGTGTATATTTA CCTCTTCGTTTGCTCAAACATGAGTGGGTATTTTTTTGTTTGTTTTTTTT TGTTGTTGTTGTTTTTGAGGCGCGTCTCACCCTGTTGCCCAGGCTGGAGT GCAATGGCGCGTTCTCTGCTCACTACAGCACCCGCTTCCCAGGTTGAAGT GATTCTCTTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGTGCCCACCA CCGCGCCCAGCTAATTTTTTAATTTTTAGTAGAGACAGGGTTTTACCATG TTGGCCAGGCTGGTCTTGAACTCCTGACCCTCAAGTGATCTGCCCACCTT GGCCTCCCTAAGTGCTGGGATTATAGGCGTGAGCCACCATGCTCAGCCAT TAAGGTATTTTGTTAAGAACTTTAAGTTTAGGGTAAGAAGAATGAAAATG ATCCAGAAAAATGCAAGCAAGTCCACATGGAGATTTGGAGGACACTGGTT AAAGAATTTATTTCTTTGTATAGTATACTATGTTCATGGTGCAGATACTA CAACATTGTGGCATTTTAGACTCGTTGAGTTTCTTGGGCACTCCCAAGGG CGTTGGGGTCATAAGGAGACTATAACTCTACAGATTGTGAATATATTTAT TTTCAAGTTGCATTCTTTGTCTTTTTAAGCAATCAGATTTCAAGAGAGCT CAAGCTTTCAGAAGTCAATGTGAAAATTCCTTCCTAGGCTGTCCCACAGT CTTTGCTGCCCTTAGATGAAGCCACTTGTTTCAAGATGACTACTTTGGGG TTGGGTTTTCATCTAAACACATTTTTCCAGTCTTATTAGATAAATTAGTC CATATGGTTGGTTAATCAAGAGCCTTCTGGGTTTGGTTTGGTGGCATTAA
ATGG SEQ ID. NO. 6 >gi|4502703|ref|NP_001245.1| CDC6 homolog [Homo sapiens] MPQTRSQAQATISFPKRKLSRALNKAKNSSDAKLEPTNVQTVTCSPRVKA LPLSPRKRLGDDNLCNTPHLPPCSPPKQGKKENGPPHSHTLKGRRLVFDN QLTIKSPSKRELAKVHQNKILSSVRKSQEITTNSEQRCPLKKESACVRLF KQEGTCYQQAKLVLNTAVPDRLPAREREMDVIRNFLREHICGKKAGSLYL SGAPGTGKTACLSRILQDLKKELKGFKTIMLNCMSLKTAQAVFPAIAQEI CQEEVSRPAGKDMMRKLEKHMTAEKGPMIVLVLDEMDQLDSKGQDVLYTL FEWPWLSNSHLVLIGIANTLDLTDRILPRLQAREKCKPQLLNFPPYTRNQ IVTILQDRLNQVSRDQVLDNAAVQFCARKVSAVSGDVRKALDVCRRAIEI VESDVKSQTILKPLSECKSPSEPLIPKRVGLIHISQVISEVDGNRMTLSQ EGAQDSFPLQQKILVCSLMLLIRQLKIKEVTLGKLYEAYSKVCRKQQVAA VDQSECLSLSGLLEARGILGLKRNKETRLTKVFFKIEEKEIEHALKDKAL IGNILATGLP SEQ ID NO. 7 >gi|4502858|ref|NM_001827.1| Homo sapiens CDC28 protein kinase regulatory subunit 2 (CKS2), mRNA AGTCTCCGGCGAGTTGTTGCCTGGGCTGGACGTGGTTTTGTCTGCTGCGC CCGCTCTTCGCGCTCTCGTTTCATTTTCTGCAGCGCGCCACGAGGATGGC CCACAAGCAGATCTACTACTCGGACAAGTACTTCGACGAACACTACGAGT ACCGGCATGTTATGTTACCCAGAGAACTTTCCAAACAAGTACCTAAAACT CATCTGATGTCTGAAGAGGAGTGGAGGAGACTTGGTGTCCAACAGAGTCT AGGCTGGGTTCATTACATGATTCATGAGCCAGAACCACATATTCTTCTCT TTAGACGACCTCTTCCAAAAGATCAACAAAAATGAAGTTTATCTGGGGAT CGTCAAATCTTTTTCAAATTTAATGTATATGTGTATATAAGGTAGTATTC AGTGAATACTTGAGAAATGTACAAATCTTTCATCCATACCTGTGCATGAG CTGTATTCTTCACAGCAACAGAGCTCAGTTAAATGCAACTGCAAGTAGGT TACTGTAAGATGTTTAAGATAAAAGTTCTTCCAGTCAGTTTTTCTCTTAA GTGCCTGTTTGAGTTTACTGAAACAGTTTACTTTTGTTCAATAAAGTTTG TATGTTGCATTTAAAAAAAAAAAAAAA SEQ ID NO. 8 >gi|4502859|ref|NP_001818.1| CDC28 protein kinase 2 [Homo sapiens] MAHKQIYYSDKYFDEHYEYRHVMLPRELSKQVPKTHLMSEEEWRRLGVQQ SLGWVHYMIHEPEPHILLFRRPLPKDQQK SEQ ID NO. 9 >gi|51468493|ref|XM_166103.5| PREDICTED: Homo sapiens DNA2 DNA replication helicase 2-like (yeast) (DNA2L), mRNA ATGAAGACTCCCTGTATTCCCAGTCCTAAGCAAGGGAGCAAAGGCAGGGG ACAGAGCCCTGCTGCTCAGGTGAGGGGCCCCACGTGGAACGCGCCGGCGC GGGAGGGGCGGCCTGGCGCAGGTCATTTGGGACATCTGCGGGTTGGCGCA TGCGCGCGAGGTGCGCAGGCTCGCGCCTTTTTCCCTTTTCTAAGCTTTCT GTGTTACCCCCGGTTCCGCTGTCTTTTCTGTCTACAGTTTGCGATCCCCG CGTCCAGGATGGAGCAGCTGAACGAACTGGAGCTGCTGATGGAGAAGAGT TTTTGGGAGGAGGCGGAGCTGCCGGCGGAGCTATTTCAGAAGAAAGTGGT AGCTTCCTTTCCAAGAACAGTTCTGAGCACAGGAATGGATAACCGGTACC TGGTGTTGGCAGTCAATACTGTACAGAACAAAGAGGGAAACTGTGAAAAG CGCCTGGTCATCACTGCTTCACAGTCACTAGAAAATAAAGAACTATGCAT CCTTAGGAATGACTGGTGTTCTGTTCCAGTAGAGCCAGGAGATATCATTC ATTTGGAGGGAGACTGCACATCTGACACTTGGATAATAGATAAAGATTTT GGATATTTGATTCTGTATCCAGACATGCTGATTTCTGGCACCAGCATAGC CAGTAGTATTCGATGTATGAGAAGAGCTGTCCTGAGTGAAACTTTTAGGA GCTCTGATCCAGCCACACGCCAAATGCTAATTGGTACGGTTCTCCATGAG GTGTTTCAAAAAGCCATAAATAATAGCTTTGCCCCAGAAAAGCTACAAGA ACTTGCTTTTCAAACAATTCAAGAAATAAGACATTTGAAGGAAATGTACC GCTTAAATCTAAGTCAAGATGAAATAAAACAAGAAGTAGAGGACTATCTT CCTTCGTTTTGTAAATGGGCAGGAGATTTCATGCATAAAAACACTTCGAC TGACTTCCCTCAGATGCAGCTCTCTCTGCCAAGTGATAATAGTAAGGATA ATTCAACATGTAACATTGAAGTCGTGAAACCAATGGATATTGAAGAAAGC ATTTGGTCCCCTAGGTTTGGATTGAAAGGCAAAATAGATGTTACAGTTGG TGTGAAAATACATCGAGGGTATAAAACAAAATACAAGATAATGCCGCTGG AACTTAAAACTGGCAAAGAATCAAATTCTATTGAACACCGTAGTCAGGTT GTTCTGTACACTCTACTAAGCCAAGAGAGAAGAGCTGATCCAGAGGCTGG CTTGCTTCTCTACCTCAAGACTGGTCAGATGTACCCTGTGCCTGCCAACC ATCTAGATAAAAGAGAATTATTAAAGCTAAGAAACCAGATGGCATTCTCA TTGTTTCACCGTATTAGCAAATCTGCTACTAGACAGAAGACACAGCTTGC TTCTTTGCCACAAATAATTGAGGAAGAGAAAACTTGTAAATATTGTTCAC AAATTGGCAATTGTGCTCTTTATAGCAGAGCAGTTGAACAACAGATGGAT TGTAGTTCAGTCCCAATTGTGATGCTGCCCAAAATAGAAGAAGAAACCCA GCATCTGAAGCAAACACACTTAGAATATTTCAGCCTTTGGTGTCTAATGT TAACCCTGGAGTCACAATCGAAGGATAATAAAAAGAATCACCAAAATATC TGGCTAATGCCTGCTTCGGAAATGGAGAAGAGTGGCAGTTGCATTGGAAA CCTGATTAGAATGGAACATGTAAAGATAGTTTGTGATGGGCAATATTTAC ATAATTTCCAATGTAAACATGGTGCCATACCTGTCACAAATCTAATGGCA GGTGACAGAGTTATTGTAAGTGGAGAAGAAAGGTCACTGTTTGCTTTGTC TAGAGGATATGTGAAGGAGATTAACATGACAACAGTAACTTGTTTATTAG ACAGAAACTTGTCGGTCCTTCCAGAATCAACTTTGTTCAGATTAGACCAA GAAGAAAAAAATTGTGATATAGATACCCCATTAGGAAATCTTTCCAAATT GATGGAAAACACGTTTGTCAGCAAAAAACTTCGAGATTTAATTATTGACT TTCGTGAACCTCAGTTTATATCCTACCTTAGTTCTGTTCTTCCACATGAT GCAAAGGATACAGTTGCCTGCATTCTAAAGGGTTTGAATAAGCCTCAGAG GCAAGCGATGAAAAAGGTACTTCTTTCAAAAGACTACACACTCATCGTGG GTATGCCTGGGACAGGAAAAACAACTACGATATGTACTCTCGTAAGAATT CTCTACGCCTGTGGTTTTAGCGTTTTGTTGACCAGCTATACACACTCTGC TGTTGACAATATTCTTTTGAAGTTAGCCAAGTTTAAAATAGGATTTTTGC GTTTGGGTCAGATTCAGAAGGTTCATCCAGCTATCCAGCAATTTACAGAG CAAGAAATTTGCAGATCAAAGTCCATTAAATCCTTAGCTCTTCTAGAAGA ACTCTACAATAGTCAACTTATAGTTGCAACAACATGTATGGGAATAAACC ATCCAATATTTTCCCGTAAAATTTTTGATTTTTGTATTGTGGATGAAGCC TCTCAAATTAGCCAACCAATTTGTCTGGGCCCCCTTTTTTTTTCACGGAG ATTTGTGTTAGTGGGGGACCATCAGCAGCTTCCTCCCCTGGTGCTAAACC GTGAAGCAAGAGCTCTTGGCATGAGTGAAAGCTTATTCAAGAGGCTGGAG CAGAATAAGAGTGCTGTTGTACAGTTAACCGTGCAGTACAGAATGAACAG TAAAATTATGTCCTTAAGTAATAAGCTGACCTATGAGGGCAAGCTGGAGT GTGGATCAGACAAAGTGGCCAATGCAGTGATAAACCTACGTCACTTTAAA GATGTGAAGCTGGAACTGGAATTTTATGCTGACTATTCTGATAATCCTTG GTTGATGGGAGTATTTGAACCCAACAATCCTGTTTGTTTCCTTAATACAG ACAAGGTTCCAGCGCCAGAACAAGTTGAAAAAGGTGGTGTGAGCAATGTA ACAGAAGCCAAACTCATAGTTTTCCTAACCTCCATTTTTGTTAAGGCTGG ATGCAGTCCCTCTGATATTGGTATTATTGCACCGTACAGGCAGCAATTAA AGATCATCAATGATTTATTGGCACGTTCTATTGGGATGGTCGAAGTTAAT ACAGTAGACAAATACCAAGGAAGGGACAAAAGTATTGTCCTAGTATCTTT TGTTAGAAGTAATAAGGATGGAACTGTTGGTGAACTCTTGAAAGATTGGC GACGTCTTAATGTTGCTATAACCAGAGCCAAACATAAACTGATTCTTCTG GGGTGTGTGCCCTCACTAAATTGCTATCCTCCTTTGGAGAAGCTGCTTAA TCATTTAAACTCAGAAAAATTAATCATTGATCTTCCATCAAGAGAACATG AAAGTCTTTGCCACATATTGGGTGACTTTCAAAGAGAATAAAACACTATT TCCCTTGCCTTTTCATACTAGGGCAGTATCTCCTCTAGCTAGTGCCCATA CAGAAAATTCTATCACCATACAAAATTTAATGCAGTATTTATGTTTTAAA GCACAGGTGTACCGAAAACTGTGAAAAGTCTGAATTTATGGGTTCTATGC ATGCATTTTTGCCTAACCTAGAGAAAGAGTTTGATAAATTTTTACCAGCT TTGAAGATGGATTAACTTTTGACTTTGAGCTTTAAACTTTTAAGTCAGAC ATTTCAGGACTAATTTGATTTTGTAGATATCATTGTAAGAACTTTATTTG AAAGACTGAATAAAGGGATTTGATTTGTTTTCATCATTTAAGCACAGTCT TGTGATGATGAGAACATAAGTGTGATTCTTTTCTGTATTTTGAGGTCCCT AATCCAAAGCCCATTTTGCTAGGATTTTTTCTGCTATCAGATGTGTTTTC ACTCTAAACCTAGTCTTTTATGACATGAATTGATTACTTCCTGTTAATTT TCTATCCTCCCTTACTATCCTCCTTTTTTGTTTTCAGTATTCAGTATTTC AGTATTCTAGAGTAGATTTTGATATAAAAGAAAATAATTCTTACATCATC TTTTGCAACAAATTTTGTTTTCTGAATTGATAATAAATTTAAAAAGTTGA TTCCTATTTTCACATATGTTCATATGCCCCTATGTTTGGGGGTATCACTC AGTTTTCCCTTTTTTGTGTAAAGATGTTTTGTAAAACAAAATTGTCTCAA AGTGATTATATTATATATATAAAAAGTAACAGATTTTAACAAAGGTTAAA AGATTCTTGGGGTAACAGATTCTTCTGGGGCTTGGAAATCTTCCATTTCT CTTGAGGGTTTTTTTTAATGAGTGTTAAATATGTTAAAATTTTTATTTCT ACCTCATGTGTTTTTTTAAATTATTACTTGAAGTTTTTTATTTAATAAAT TTTTTCTACTAATGG SEQ ID NO. 10 >gi|51468494|ref|XP_166103.4| PREDICTED: DNA2 DNA replication helicase 2-like [Homo sapiens] MKTPCIPSPKQGSKGRGQSPAAQVRGPTWNAPAREGRPGAGHLGHLRVGA
CARGAQARAFFPFLSFLCYPRFRCLFCLQFAIPASRMEQLNELELLMEKS FWEEAELPAELFQKKVVASFPRTVLSTGMDNRYLVLAVNTVQNKEGNCEK RLVITASQSLENKELCILRNDWCSVPVEPGDIIHLEGDCTSDTWIIDKDF GYLILYPDMLISGTSIASSIRCMRRAVLSETFRSSDPATRQMLIGTVLHE VFQKAINNSFAPEKLQELAFQTIQEIRHLKEMYRLNLSQDEIKQEVEDYL PSFCKWAGDFMHKNTSTDFPQMQLSLPSDNSKDNSTCNIEVVKPMDIEES IWSPRFGLKGkIDVTVGVKIHRGYKTKYKIMPLELKTGKESNSIEHRSQV VLYTLLSQERRADPEAGLLLYLKTGQMYPVPANHLDKRELLKLRNQMAFS LFHRISKSATRQKTQLASLPQIIEEEKTCKYCSQIGNCALYSRAVEQQMD CSSVPIVMLPKIEEETQHLKQTHLEYFSLWCLMLTLESQSKDNKKNHQNI WLMPASEMEKSGSCIGNLIRMEHVKIVCDGQYLHNFQCKHGAIPVTNLMA GDRVIVSGEERSLFALSRGYVKEINMTTVTCLLDRNLSVLPESTLFRLDQ EEKNCDIDTPLGNLSKLMENTFVSKKLRDLIIDFREPQFISYLSSVLPHD AKDTVACILKGLNKPQRQAMKKVLLSKDYTLIVGMPGTGKTTTICTLVRI LYACGFSVLLTSYTHSAVDNILLKLAKFKIGFLRLGQIQKVHPAIQQFTE QEICRSKSIKSLALLEELYNSQLIVATTCMGINHPIFSRKIFDFCIVDEA SQISQPICLGPLFFSRRFVLVGDHQQLPPLVLNREARALGMSESLFKRLE QNKSAVVQLTVQYRMNSKIMSLSNKLTYEGKLECGSDKVANAVINLRHFK DVKLELEFYADYSDNPWLMGVFEPNNPVCFLNTDKVPAPEQVEKGGVSNV TEAKLIVFLTSIFVKAGCSPSDIGIIAPYRQQLKIINDLLARSIGMVEVN TVDKYQGRDKSIVLVSFVRSNKDGTVGELLKDWRRLNVAITRAKHKLILL GCVPSLNCYPPLEKLLNHLNSEKLIIDLPSREHESLCHILGDFQRE
[0082]In one embodiment, the level of other markers other than CDC2, CDC6, DNA2L or CKS2 may also be determined. For example, the level of ER-β may also be determined.
[0083]A "marker nucleic acid" includes a nucleic acid (e.g., mRNA, cDNA) encoded by or corresponding to a marker of the invention. Such marker nucleic acids include DNA (e.g., genomic DNA or cDNA) comprising the entire or a partial sequence of the marker or the complement of such a sequence. The marker nucleic acids also include RNA comprising the entire or a partial sequence of the marker or the complement of such a sequence.
[0084]A "marker protein" includes a protein encoded by or corresponding to a marker of the invention. A marker protein may comprise the entire or a partial sequence of the marker or a variant of the marker. For the avoidance of doubt, the terms "protein" and "polypeptide" are used interchangeably herein.
[0085]Thus, by determining the level of a particular marker, the level of one or more of the following moieties may, for example, be determined: full-length cDNA markers, full-length genomic DNA markers, fragments of the full-length cDNA markers or genomic DNA markers, and transcribed polynucleotides.
[0086]The level of a marker may be determined by any means known in the art. The level may be determined by, for example, determining the level of nucleic acid transcribed from a marker gene. Alternatively, or additionally, the level of specific proteins translated from mRNA transcribed from a marker genie may be determined. In yet another embodiment, the level of a metabolite which is produced directly (i.e., catalyzed) or indirectly or "consumed" by the corresponding marker protein could be determined.
[0087]An exemplary assay for determining the level of a marker involves obtaining a sample (e.g., a breast associated body fluid) of an individual and contacting the sample with a probe (e.g. antibody, oligonucleotide) capable of detecting the marker protein or marker nucleic acid (e.g., mRNA, genomic DNA, or cDNA) under appropriate conditions and for a time sufficient allow the marker and probe to interact and bind, thus forming a complex that can be removed and/or detected in the reaction mixture. The detection methods of the invention can thus be used to detect mRNA, protein, cDNA, or genomic DNA, for example, in a biological sample in vitro as well as in vivo.
[0088]These assays can be conducted in a variety of way. For example, one method to conduct such an assay would involve anchoring the marker or probe onto a solid support and detecting target marker/probe complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, a sample of an individual, which is to be assayed for presence, amount and/or concentration of marker, can be anchored onto a solid support. In another embodiment, the reverse situation is possible, in which the probe can be anchored to a solid support (e.g. a nylon membrane or a chip) and a sample of an individual can be allowed to react as an unanchored component of the assay. In one embodiment, the probes may be immobilized on a microarray.
[0089]In order to conduct assays with the above-mentioned approaches, the non-immobilized component is added to the solid support upon which the second component is anchored. After the reaction is complete, uncomplexed components may be removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized upon the solid support. The detection of marker/probe complexes anchored to the solid support can be accomplished in a number of methods.
[0090]In a preferred embodiment, the probe, when it is the unanchored assay component, can be labeled for the purpose of detection and readout of the assay, either directly or indirectly, with a detectable label. It is also possible to directly detect marker/probe complex formation without further manipulation or labeling of either component (marker or probe), for example by utilizing the technique of fluorescence energy transfer (see; for example, Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No. 4,868,103).
[0091]The level of expression of specific marker genes can, for example, be accomplished by determining the amount of mRNA (or polynucleotides derived therefrom) present in a sample. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by a marker of the present invention.
[0092]Many techniques for the detection and quantification of mRNA levels involve contacting the mRNA with a nucleic acid molecule probe) that can hybridize to the mRNA (or polynucleotide derived therefrom). The nucleic acid probe can be, for example, a polynucleotide of at least 7, 10, 15, 17, 18, 20, 25, 30, 40, 50, 100, 500, nucleotide residues in length. Probes may include, but are not limited to, oligonucleotides, cDNA, or RNA. Probes may contain a detectable label, such as a fluorescent or chemiluminescent label. When a method of assessing marker expression is used which involves hybridization of one nucleic acid with another, it is preferred that the hybridization be performed under stringent hybridization conditions.
[0093]Methods for conducting polynucleotide hybridization assays have been well developed in the art. Hybridization assay procedures and conditions will vary depending on the application and are selected in accordance with the general binding methods known including those referred to in: Maniatis et al. Molecular Cloning: A Laboratory Manual (2nd Ed. Cold Spring Harbor; N.Y, 1989); Berger and Kimmel Methods in Enzymology, Vol, 152, Guide to Molecular Cloning Techniques (Academic Press, Inc., San Diego, Calif., 1987); Young and Davis, P.N.A.S, 80: 1194 (1983). Methods and apparatuss for carrying out repeated and controlled hybridization reactions have been described in U.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,613 each of which are incorporated herein by reference.
[0094]The present invention contemplates signal detection of hybridization between ligands in certain preferred embodiments. See U.S. Pat. Nos. 5,143,854; 5,578,832; 5,631,734; 5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and 6,215,625, U.S. Patent Application No. 60/364,731 and PCT Application No. PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.
[0095]Methods and apparatus for signal detection and processing of intensity data are disclosed in for example, U.S. Pat. Nos. 5,143,854, 5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S. Patent Application 60/364,731 and in PCT Application PCT/US99/06097 (published as WO99/47964), each of which also is hereby incorporated by reference in its entirety for all purposes.
[0096]Any method for determining nucleic acid or protein levels can be used in the present invention and the examples described herein are not intended to be limiting.
[0097]In one format, mRNA is immobilized on a solid support and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a solid support such as a filter. Nucleic acid probes representing one or more markers are then hybridized to the filter by northern hybridization, and the amount of marker-derived RNA is determined. Such determination can be visual, or machine-aided, for example, by use of a densitometer. In an alternative format, the probe(s) are immobilized on a solid support and the nucleic acid is contacted with the probe(s), for example, in an Affymetrix gene chip array. The present invention also contemplates sample preparation methods in certain preferred embodiments. For example, prior to or concurrent with gene expression analysis, the sample may be amplified by a variety of mechanisms, some of which may employ amplification techniques such as PCR. See, e.g., PCR Technology: Principles and Applications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, CA, 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202; 4,683,195, 4,800,159 4,965,183, and 5,333,675, and each of which is incorporated herein by reference in their entireties for all purposes. The sample may be amplified on the array. See, for example, U.S. Pat. No. 6,300,070 and U.S. patent application Ser. No. 09/513,300, which are incorporated herein by reference.
[0098]Other suitable amplification methods include the ligase chain reaction (LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988) and Barringer et at. Gene 89:117 (1990)), transcription-amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989) and WO88/10315), self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) and WO90/06995), selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245) and nucleic acid based sequence amplification (NABSA). (See, U.S. Pat. No. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporated herein by reference). Other amplification methods that may be used are described in, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317, each of which is incorporated herein by reference.
[0099]Additional methods of sample preparation and techniques for reducing the complexity of a nucleic sample are described in Dong et al., Genome Research 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 and U.S. patent application Ser. Nos. 09/916,135, 09/920,491, 09/910,292, and 10/013,598.
[0100]In one embodiment of the present invention, the level of a marker protein is determined. A preferred agent for determining the level of a marker protein of the invention is an antibody capable of binding to such a protein or a fragment thereof, preferably an antibody with a detectable label.
[0101]Suitable antibodies can be produced using techniques well known to those of skill in the art and disclosed in, for example, U.S. Pat. Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745. Preferably, monoclonal antibodies are employed. Monoclonal antibodies are generally prepared using the method of Kohler & Milstein (1975) Nature 256:495-497, or a modification thereof. Typically, a mouse or rat is immunized with the protein of interest. Rabbits may also be used. The spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of non-specifically adherent cells) by applying a cell suspension to a plate or well coated with the antigen. B-cells, expressing membrane-bound immunoglobulin specific for the antigen, will, bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, "HAT"). The resulting hybridomas are plated by limiting dilution; and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected monoclonal antibody-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (e.g., as ascites in mice).
[0102]A variety of formats can be employed, to determine whether a sample contains a protein that binds to a given antibody. Examples of such formats include, but are not limited to, enzyme immunoassay (EIA), radioimmunoassay (RIA), enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitation and immunofluorescence. In such uses, it is generally preferable to immobilize either the antibody or proteins on a solid support. Suitable supports include any support capable of binding an antigen or an, antibody. In one embodiment, marker-derived protein levels can be determined by constructing an antibody microarray in which binding sites comprise immobilized, preferably monoclonal, antibodies specific to a marker protein. By utilising antibodies which are specific for different marker proteins, the level of more than one marker protein may be determined using a single microarray.
[0103]In addition, preferred in vivo techniques for detection of a marker protein include introducing into a subject a labeled antibody directed against a marker protein. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
[0104]The present invention can employ solid supports, including arrays in some preferred embodiments. Methods and techniques applicable to polymer (including protein) array synthesis have been described in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730 (International Publication Number WO 99/36760) and PCT/US01/04285, which are all incorporated herein by reference in their entirety for all purposes.
[0105]Patents that describe synthesis techniques in specific embodiments include U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189, 5,889,165, and 5,959,098. Nucleic acid arrays are described in many of the above patents, but the same techniques may be applied to polypeptide (e.g. antibody) arrays.
[0106]In preferred embodiments, polynucleotide microarrays are used to determine the level of a marker. In this way, the expression status of more than one marker may be assessed simultaneously. In a specific embodiment, the invention provides for arrays comprising probes hybridizable to at least one, two or three of (and preferably to all four of) CDC2, CDC6, CKS2 and DNA2L. The microarrays may comprise further probes suitable for detecting one or more markers additional to CDC2, CDC6, CKS2 or DNA2L, e.g. for detecting ER-β.
[0107]Microarrays may be prepared by selecting probes which comprise a polynucleotide sequence, and then immobilizing such probes to a solid support or surface. For example, the probes may comprise DNA sequences, RNA sequences, or copolymer sequences of DNA and RNA. The polynucleotide sequences of the probes may also comprise DNA and/or RNA analogues, or combinations thereof. For example, the polynucleotide sequences of the probes may be full or partial fragments of genomic DNA. The polynucleotide sequences of the probes may also be synthesized nucleotide sequences, such as synthetic oligonucleotide sequences. The probe sequences can be synthesized either enzymatically in vivo, enzymatically in vitro (eg., by PCR), or nonenzymatically in vitro.
[0108]The probe or probes used in the methods of the invention are preferably immobilized to a solid support which may be either porous or non-porous. For example, the probes of the invention may be polynucleotide sequences which are attached to a nitrocellulose or nylon membrane or filter covalently at either the 3' or the 5' end of the polynucleotide. Alternatively, the solid support may be a glass or plastic surface. In a particularly preferred embodiment, hybridization levels are measured to microarrays of probes consisting of a solid support on the surface of which are immobilized a population of polynucleotides, such as a population of DNA or DNA mimics, or, alternatively, a population of RNA or RNA mimics. The solid support may be a nonporous or, optionally, a porous material such as a gel.
[0109]A skilled artisan will also appreciate that it may be desirable to include positive control probes, e.g., probes known to be complementary and hybridizable to sequences in the target polynucleotide molecules, and/or negative control probes, e.g., probes known to not be complementary and hybridizable to sequences in the target polynucleotide molecules, on the array.
[0110]The present invention may also make use of the several embodiments of the array or arrays and the processing described in U.S. Pat. Nos. 5,545,531 and 5,874,219. These patents are incorporated herein by reference in their entireties for all purposes.
[0111]The present invention may make use of various program products and software for a variety of purposes, such as probe design, management of data, analysis, and instrument operation. See, U.S. Pat. Nos. 5,795,716; 5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170.
[0112]In some embodiments, the expression levels of marker nucleic acids may be used to generate expression profiles. An expression profile of a particular sample is essentially a "fingerprint" of the state of the sample--while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is characteristic of the state of the cell. This allows normal tissue to be distinguished from, for example, cancerous tissue, or cancer tissue to be compared with tissue from surviving cancer patients. Comparing expression profiles in different cancer states identifies genes (e.g. up- and down-regulated genes) that are important in each of these states. Molecular profiling may distinguish subtypes of a currently collective disease designation, e.g., different forms or stages of a cancer. Metastatic tissue may also be analyzed to determine the stage of cancer in the tissue, or origin of primary tumor, e.g., metastasis from a remote primary site.
[0113]By assessing the level of one or more of the markers of the invention (i.e. CDC2, CDC6, DNA2L and CKS2) information may be gleaned concerning ER-β function. As mentioned above, the level of the markers of the invention have been correlated with clinical outcome and as discussed in the Examples section below, the level of the markers have been found to correlate with the following: (i) tumor grade (grade 1, 2, or 3); (ii) disease recurrence within 10 years of surgery; (iii) death within 10 years of surgery; and (iv) and p53 mutation status.
[0114]Since the levels of CDC2, CDC6, DNA2L and CKS2 are inversely correlated with ER-β function then a low level of one or more of these markers is indicative of a good prognosis. A low level of one or more of these markers is also indicative of the patient having a good response to adjuvant endocrine therapy and thus measurement of one or more of the markers of the invention may be used in treatment selection.
[0115]In addition to determining the level of a marker of the invention, other markers (e.g. a marker as included in Table 3) and other factors may also be taken into account e.g. gender, age, previous cancer history, benign breast disease, hereditary factors (family history of cancer), early age at menarche (first menstrual period), late age at menopause, late age at first full-term pregnancy, obesity, low physical activity, use of postmenopausal hormone replacement therapy, use of oral contraceptives, exposure to ionizing radiation, dietary practices, or alcohol consumption.
[0116]The methods, arrays, kits etc. of the invention may be used to evaluate a patient before, during and after treatment, for example, to evaluate the reduction in tumor burden. The methods of the invention may be employed in vivo or in vitro. The methods of the invention may employ a kit or array as described herein.
[0117]It is envisaged that the invention will find utility in at least the following areas: [0118]Prognosis [0119]Characterization of the cancer (e.g. stage, grade, or ER-β level) [0120]Treatment selection [0121]Assessment of treatment efficacy [0122]Assessment of cancer progression of regression [0123]Identification and development of new or existing treatments for cancer [0124]Assessing the carcinogenic potential of a test compound
[0125]The invention is envisaged as finding particular utility with respect to breast cancer but may also find utility with respect to other cancers. Thus, the cancer may for example be adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma or cancers of the blood, bone, bone marrow, brain, breast, gastrointestinal tract (esophagus, stomach, small intestine or colon), heart, kidney, liver, lung, lymph, muscle, nerve, ovary, pancreas, prostate, skin, spleen, testis, or uterus. In one embodiment, the cancer is an endocrine-related cancer. In one embodiment, the cancer is a primary cancer. In one embodiment the cancer is breast cancer. "Breast cancer" as used herein includes carcinomas, (e.g., carcinoma in situ, invasive carcinoma, metastatic carcinoma) and pre-malignant conditions. In one embodiment, the cancer is a primary breast cancer. In one embodiment the cancer is ER-α positive. The terms tumor and cancer and used interchangeably herein unless the context indicates otherwise.
[0126]It is envisaged that the present invention will find particular utility in relation to ER-α positive tumors and in the area of adjuvant endocrine hormone therapy. In particular, it is envisaged that the invention will find particular utility in respect of patients selected for adjuvant endocrine hormone therapy (e.g. patients which are receiving or which have previously received adjuvant endocrine hormone therapy or which have been identified as patients for which adjuvant endocrine hormone therapy would be appropriate for).
[0127]In one embodiment of the invention, the level of one or more of the markers of the invention is used in predicting the prognosis of an individual. The level of one or more of the markers may provide information on, inter alia, patient survival, likelihood of disease recurrence and disease metastasis (patient survival, disease recurrence and disease metastasis may be assessed in relation to a defined timepoint, e.g. at a given number of years after cancer surgery (e.g. surgery to remove one or more tumors) or after initial diagnosis). In one embodiment, the prognosis comprises a prediction of DSS or DFS.
[0128]Depending on the level of the one or markers of the invention in the patient sample, individual appropriate treatment may be selected for the patient. For instance, the continuation of existing treatment may be deemed appropriate and/or the commencement of additional or a different treatment for that individual may be deemed appropriate.
[0129]The markers of the invention may be useful as pharmacogenomic markers. As used herein, a "pharmacogenomic marker" is an objective biochemical marker whose expression level correlates with a specific clinical drug response or other treatment response or susceptibility in an individual. The presence or quantity of the pharmacogenomic marker expression is related to the predicted response of the patient and more particularly the patient's tumor to treatment, e.g. with a specific drug or class of drugs, e.g. adjuvant endocrine therapy. By assessing the presence or quantity of the expression of one or more pharmacogenomic markers in a patient, a treatment which is appropriate for the patient may be selected.
[0130]For example, based on the level of marker of the invention, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the patient. The use of a pharmacogenomic marker therefore permits selecting or designing the most appropriate treatment for each cancer patient without trying different drugs or treatment regimes.
[0131]Knowledge of the level of a marker of the invention (optionally in conjunction with other factors e.g. cell morphology) may be used to aid in the characterization of a cell/tumor, for instance its ER-β level and/or the grade of the tumor (e.g. grade 1, 2, 3 according to the Scarff-Bloom-Richardson grade system). Information regarding the characterization of a cell/tumor may for example be used in the prognosis of the patient and/or the selection of appropriate treatment for the patient.
[0132]Doctors use tumor grade and many other factors, such as cancer stage, to develop an individual treatment plan for the patient and to predict the patient's prognosis. Generally, a lower grade indicates a better prognosis (the likely outcome or course of a disease; the chance of recovery or recurrence).
[0133]In one embodiment, the level of one or more markers of the invention is used in the characterization of a tumor according to the Elston-Ellis modification of the Scarff-Bloom-Richardson grade system. Briefly, according to the Scarff-Bloom-Richardson grade system there are the following grades: [0134]Grade 1 (lowest): Well-differentiated breast cells; cells generally appear normal and are not growing rapidly; cancer arranged in small tubules. 5 years survival: 95%; 7 year survival: 90%; [0135]Grade 2 Moderately differentiated breast cells; have characteristics between Grade 1 and Grade 3 tumors. 5 years survival 75%; 7 year survival: 63%; [0136]Grade 3 (highest). Poorly-differentiated breast cells; cells do not appear normal and tend to grow and spread more aggressively; 5 years survival: 50%; 7 year survival 45%.
[0137]The Elston-Ellis modification provides a somewhat more objective criteria for the three component elements of grading and specifically addresses mitosis, counting in a more rigorous fashion. For example hyperchromatic nuclei and apoptotic cells which are counted in the original SBR system stem are excluded in the Elston-Ellis modification and the area being assessed is specifically defined in square millimeters. These modifications have enhanced reproducibility of grading among pathologists and to a considerable extent have fostered acceptance of grading by clinicians (Elston, C. W. and Ellis I. O. (1991) Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology, 19, 403-10.
[0138]In one embodiment of the invention, the level, of one or more markers of the invention is used in the staging of a tumour. Staging describes the extent or severity of an individual's cancer based on the extent of the original (primary) tumor and the extent of spread in the body. Staging is important as it helps the doctor plan a person's treatment and the stage can be used to estimate the person's prognosis (likely outcome or course of the disease).
[0139]The common elements considered in most staging systems are: location of the primary tumor; tumor size and number of tumors, lymph node involvement (spread of cancer into lymph nodes), cell type and tumor grade and presence or absence of metastasis.
[0140]The TNM system is one of the most commonly used, staging systems. The TNM system is based on the extent of the tumor (T), the extent of spread to the lymph nodes (N), and the presence of metastasis (M). A number is added to each letter to indicate the size or extent of the tumor and the extent of spread.
[0141]In one embodiment of the invention, the knowledge of the level of a marker of the invention may be used to decide whether a patient should be treated with adjuvant endocrine therapy (e.g. tamoxifen).
[0142]The level of a maker of the invention may be measured at one or more time points. By monitoring the level of one or more of the markers of the invention, cancer progression (cancer progression as used herein includes cancer growth and metastasis), cancer regression and treatment efficacy may be assessed. The progression or regression of a cancer or the efficacy of a treatment may for example be assessed by using the grading or staging system as discussed above.
[0143]In one embodiment of the invention, the level of a marker of the invention may be used to help identify or develop a new or existing treatment for cancer. For example, gene expression profiles can allow screening of drug candidates with the goal of mimicking or altering a particular expression profile. This may be done by designing a microarray (for example) comprising one or more probes capable of hybridizing with at least one marker nucleic acid of the invention. The microarray can then be used to screen drug candidates that alter the expression of CDC2, CDC6, DNA2L and/or CKS2 (and optionally also ER-β). Screening assays may also be carried out at the protein level, i.e. the effect of a candidate agent on the level of one or more marker proteins of the present invention may be determined.
[0144]In a preferred embodiment, gene expression profiles are used, preferably in conjunction with high throughput screening techniques, to allow monitoring for expression profile genes after treatment with a candidate agent, for example.
[0145]Assessing the influence of agents (e.g., drug compounds) on the level of expression of a marker of the invention can be applied not only in basic drug screening or assessing the efficacy of treatment in an individual, but also in clinical trials. For example, the effectiveness of an agent to affect marker expression can be monitored in clinical trials of subjects receiving cancer treatment. In one embodiment, the present invention provides, a method for assessing the effectiveness of treatment of a subject with a candidate agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) comprising the steps of obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of one or more selected markers of the invention in the pre-administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression of the marker(s) in the post-administration samples; (v) comparing the level of expression of the marker(s) in the pre-administration sample with the level of expression of the marker(s) in the post-administration sample or samples; and optionally (vi) altering the administration of the agent to the subject accordingly. For example, increased expression of the marker(s) during the course of treatment may indicate ineffective dosage and the desirability of increasing the dosage. Conversely, decreased expression of the marker(s) may indicate efficacious treatment and no need to change dosage. In an alternative embodiment, the level of expression of the marker(s) in the post-administration samples (step iv) may be detected at multiple time points. The multiple time points may be evenly spaced or non-evenly spaced or they may be chosen randomly.
[0146]In one embodiment, the influence of a candidate agent or a known drug on marker expression may be determined by comparing the level of one or more markers of the invention in one or more patients who have received the candidate agent or known drug, with the level of the one or more markers in one or more patients who have not received the candidate agent or known drug.
[0147]The invention additionally provides a method for assessing progression or regression of cancer in a patient, the method comprising: determining the level of at least one marker of the invention in a sample from the patient; repeating the marker level assessment at a subsequent time point; and comparing the level of the marker(s) in the first and second steps, thereby assessing the progression or regression of cancer in the patient. A significantly higher level of expression of the marker in the sample at the subsequent time point from that of the sample at the first time point is an indication that the cancer has worsened in the patient, whereas a significantly lower level of expression is an indication that the breast cancer has regressed. In one embodiment, the patient has undergone surgery to remove a tumor between the first point in time and the subsequent point in time. In an alternative embodiment, the subsequent time point may comprise multiple time points, i.e. the detection of expression step may be carried out at multiple time points until such a point in time when the difference in the level of expression of the marker between the first time point and the subsequent time point is significant.
[0148]In one embodiment of the invention there is provided a method which comprises comparing expression of at least one marker of the invention in a first sample (e.g. breast cell sample) and maintained in the presence of a candidate agent and expression of the at least one marker in a second sample (e.g. breast cell sample) and maintained in the absence of the candidate agent. A significantly reduced expression of the marker(s) of the invention in the presence of the candidate agent is an indication that the candidate agent inhibits cancer. Cell samples may be used which may for example, be aliquots of single sample of normal cells obtained from a patient, pooled samples of normal cells obtained from a patient, cells of a normal cell line, aliquots of a single sample of cancer cells obtained from a patient, pooled samples of cancer cells obtained from a patient, cells of a cancer cell line, or the like. In one embodiment, the samples are cancer cells obtained from a patient and one or more of a plurality of agents known to be effective for inhibiting cancer are tested in order to identify the agent which is likely to best inhibit the cancer in the patient.
[0149]The level of the one or more markers of the invention may be compared with a control for the one or more of said markers.
[0150]In one embodiment, the marker level is compared to the level of the marker from a control, wherein the control comprises one or more tumor samples (e.g. breast cancer samples) taken from one or more patients determined as having a good prognosis ("good prognosis" control) or a poor prognosis ("poor prognosis" control), or both. Alternatively or additionally, the control may comprise one or more tumor samples (e.g. breast cancer samples) taken from one or more patients determined as being of a particular tumor type (e.g. grade, stage, p53 mutation status).
[0151]A good or bad prognosis may, for example, be assessed in terms of patient survival, likelihood of disease recurrence or disease metastasis (patient survival, disease recurrence and metastasis may for example be assessed in relation to a defined timepoint, e.g. at a given number of years after cancer surgery (e.g. surgery to remove one or more tumors) or after initial diagnosis). In one embodiment, a good or bad prognosis may be assessed in terms of DSS or DFS.
[0152]The control may comprise data obtained at the same time (e.g. in the same hybridization experiment) as the patient's individual data; or may be a stored value or set of values e.g. stored on a computer, or on computer-readable media. If the latter is used, new patient data for the selected marker(s), obtained from initial or follow-up samples, can be compared to the stored data for the same marker(s) without the need for additional control experiment.
[0153]In one aspect of the invention there is provided a kit. The kit comprises a reagent for use in determining the level of at least one marker of the invention. The kit may be promoted, distributed, or sold as a unit for performing a method of the present invention.
[0154]The kit can comprise a labeled compound or agent capable of detecting a marker protein or nucleic acid in a sample and means for determining the level of the marker protein or marker nucleic acid in the sample (e.g., an antibody which binds the protein or fragment thereof, or an oligonucleotide probe which binds to DNA or mRNA encoding the protein). Kits can also include instructions for interpreting the results obtained using the kit.
[0155]For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a marker protein; and, optionally, (2) a second, different antibody which binds to either the marker protein or the first antibody and which is optionally conjugated to a detectable label.
[0156]For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid marker and/or (2) a pair of primers useful for amplifying a marker nucleic acid molecule. The kit can also comprise, e.g., one or more of the following: a buffering agent, a preservative, or a protein stabilizing agent. The kit can further comprise one or more components for use in detecting the detectable label (e.g., an enzyme or a substrate).
[0157]The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.
[0158]It will be appreciated that the kits of the present invention may also include antibodies or oligonucleotide probes which bind to known cancer markers for example known breast cancer markers.
[0159]The kit of the invention may optionally comprise additional components useful for performing a method of the invention. By way of example, the kit may comprise fluids (e.g., SSC buffer) suitable for annealing complementary nucleic acids or for binding an antibody with a protein with which it specifically binds, one or more sample compartments, an instructional material which describes performance of a method of the invention, and the like.
[0160]In one embodiment, the kit may comprise a microarray, e.g. an oligonucleotide microarray or an antibody microarray. The microarray may be capable of being used to determine the level of one, two, three or four of CDC2, CDC6, DNA2L and CKS2 and optionally also ER-β.
[0161]In one embodiment the kit comprises software, for example software for predicting prognostic outcome or for selecting patient treatment. Such software might include instructions for the computer system's processor to receive data structures that include the level of expression of one, two, three or four of the markers of the invention (and optionally one or more further markers) in a sample (e.g. in a breast cancer tumor sample obtained from a breast cancer patient) and optionally also clinical information about the patient, e.g. the patient's age, stage of breast cancer, estrogen receptor status, and lymph node status.
[0162]The invention additionally provides a test method of assessing the carcinogenic potential of a test compound. This method may comprise the steps of: maintaining separate aliquots of cancer cells in the presence and absence of a test compound; and comparing expression of a marker of the invention in each of the aliquots. A significantly higher level of expression of the marker in the aliquot maintained in the presence of the compound, relative to that of the aliquot maintained in the absence of the compound, is an indication that the compound possesses carcinogenic potential. Expression of a marker of the invention in an aliquot may also be measured at two or more time points to determine the time required for the compound to effect its carcinogenic potential.
[0163]In an alternative embodiment, the level of a marker of the invention may be determined at least two time points in a given sample. In this way, increased or decreased expression of a marker of the invention would indicate the ability of the test compound to promote or inhibit respectively, the cancer.
EXAMPLES
[0164]Reference will now be made in detail to exemplary embodiments of the invention. While the invention will be described in conjunction with the exemplary embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover alternatives, modifications and equivalents, which may be included within the spirit and scope of the invention.
[0165]ERα and ERβ bind to the same response element but are associated with different growth effects. In order to assess distinct signatures that can discern the biological differences, the global transcriptional effect of ERβ expression on ERα action was examined.
[0166]In order to characterize the effect of ERβ expression on hormone response in ERα-positive breast tumor cells and to decipher the functions of α/β heterodimers, T-47D (ERα+/ERβ-) cells were stably transfected with an inducible ERβ expression construct to generate subline T-47 Dbeta. Using high-density DNA microarrays under conditions that induce ERβ expression and following hormone treatment, the transcriptional effects of the ERα/β coexpression was assessed. It has been shown that ERβ over-expression (induced T47 Dbeta) in this cell line inhibits E2 induced cell proliferation and downregulates the expression and activity of the cell cycle machinery (32).
[0167]In the present work, genome-wide expression profiles of genes responsive to ERβ expression and estrogen treatment were generated. Hierarchical clustering of the profiles revealed potential mechanisms of interaction between ERα and ERβ in regulating hormone response, and the potential roles of putative ERβ-modulated genes in the biology of primary breast tumors.
[0168]Paradoxically, it was found that induction of ERβ exhibited a highly similar transcriptional response as to the exposure of these ERα-positive cells to estrogen. However, detailed analysis revealed a subset of 14 DNA replication and cell cycle genes specifically down-regulated by ERβ and tightly associated with attenuation of cell proliferation. In order to determine the in vivo relevance of this inhibitory mechanism, the expression characteristics of these genes were examined in 69 ERα-positive tumors from patients who received adjuvant endocrine therapy. Association of the expression characteristics of these genes with ERβ transcript levels, clinical parameters and disease outcome was also assessed. Expression profiles of four ERβ-regulated genes, CDC2, CDC6, CKS2, and DNA2L were significantly associated with ERβ transcript levels, and the directionality is consistent with in vitro observations. Clustering of tumor samples using the expression profiles of the ERβ-associated expression cassette indicated statistically significant association with tumor grade (p=0.00934) but not with lymph node status. This suggests a primary effect of ER-β on tumor growth rather than on the metastatic phenotype.
[0169]Kaplan-Meier analysis indicated better disease outcome for the patient group with an expression signature linked to low ERβ expression cassette as compared to the high ERβ cassette group for both disease-free survival (p=0.00165) and disease-specific survival (p=0.0268). These clinical findings were further validated in an independent cohort of 45 ERα-positive tumors and appear to explain the previous survival heterogeneity of this ER positive group.
Materials and Methods
[0170]Cell culture. T-47D cells were cultured in a mixture of DMEM and Hams F12 1:1 supplemented with 5% fetal bovine serum (FBS), penicillin and streptomycim. For experiments using 17β-estradiol, DMEM without phenol red and FBS treated with dextran coated charcoal were used.
[0171]Transfection and plasmids. T-47D cells stably transfected with tetracyclin regulated ERβ expression plasmid were generated in two steps: 1) the cells were first transfected with pTet-tTAk (Gibco/Invitrogen, Carlsbad, Calif., USA) modified to contain puromycin resistance using lipofectin according to manufacturer's instructions (Gibco/Invitrogen, Carlsbad, Calif., USA). Selection was performed with 0.5 μg/ml of puromycin in the presense of 1 μg/ml of tetracycline. A good clone showing high level of induction and low basal activity was selected using the pUHC13-3 control plasmid (Gibco/Invitrogen, Carlsbad, Calif., USA). The short form of ERβ encoding 485 amino acids was fused to the flag tag (ERβ 485) and cloned into PBI-EGFP (Clontech/BD Biosciences, Palo Alto, Calif., USA) 2) This construct was then transfected into the previously described inducible clone together with a neomycin resistance plasmid. The selection was performed using 500 μg/ml of G418 (Calbiochem/EMD Biosciences, San Diego, Calif., USA).
[0172]Preparation of RNA. Cells were added, to 150 mm plates at a confluency of 40%; after one day, the normal medium was replaced by the medium described above, supplemented with 5% dextran coated charcoal treated FBS (DCCFBS). After 24 h, 10 nM ICI 182,780 was added to the cultures and incubation proceeded for an additional 48 h. For expression of ERβ, tetracycline was removed 12 hours before initiation of treatment with 17β-estradiol. At time 0 h the medium was changed to 0.5% DCCFBS and 10 nM of 17β-estradiol was added. RNA was prepared by adding 10 ml of Trizol. (Invitrogen, Carlsbad, Calif., USA) to each 150 mm plate at different time points after start of treatment and RNA was prepared according to the manufacturer's instructions.
[0173]Microarray Analysis. Microarrays were generated by spotting the Compugen (Jamesburg, N.J., USA) 19K human oligo library, made by Sigma-Genosys (The Woodlands, TX, USA), on poly-l-lysine-coated glass slides. 25 μg each of sample total RNA and human universal reference RNA (Stratagene, La Jolla, Calif., USA) were labeled with Cy5-conjugated dUTP and Cy3-conjugated dUTP (PerkinElmer, Boston, Mass., USA); respectively, and hybridized to the arrays using protocols established by the Patrick O. Brown Laboratory (Stanford University, USA) accessible at http://cmgm.stanford.edu/pbrown/protocols/index.html. Array images and data were obtained and processed using the GenePix4000B scanner and GenePix Pro software (Axon Instruments, Union City, Calif., USA). Differentially expressed genes were determined using t-test and fold-difference cut-off at multiple time points and clustered and visualized using the Eisen Cluster and TreeView programs (Eisen et al., 1998). Gene ontology of responsive genes was derived from annotations made by Compugen.
[0174]Real-time relative quantification PCR. For validation of microarray data RNA samples from the 30 hour time point were converted to cDNA using the PowerScript® Reverse Transcriptase (Clontech, CA, USA), following the recommended protocol with 1 μg of total RNA. Quantification of transcript levels was performed using SYBR Green chemistry on the ABI PRISM® 7900HT Sequence Detection System (Applied Biosystems. CA, USA). PCR amplification was carried out in 20 μl of reaction mixture consisting of 50 nM of primer pairs. The following primers were used for PCR reactions: CDC2-CCCAAATGGAAACCAGGAAG (forward) (SEQ ID NO. 12), CGTTTGGCTGGATCATAGAT (reverse) (SEQ ID NO. 13); CDC6-GGAAACCCGTTTGACAAAGG (forward) (SEQ ID NO 14), CGGGTGGGGTGTAAGAGAAGAATT (reverse) (SEQ ID NO. 15); CKS2-CTGATGTCTGAAGAGGAGTG (forward) (SEQ ID NO. 16), GGAAGAGGTCGTCTAAAGAG (reverse) (SEQ ID NO. 17); DNA2L-GGATGGAACTGTTGGTGAAC (forward) (SEQ. ID NO. 18), ATTTAGTGAGGGCACACACC (reverse) (SEQ ID NO. 19); ERβ-TCCATGCGCCTGGCTAAC (forward) (SEQ ID NO. 20), CAGATGTTCCATGCCCTTGTTA (reverse) (SEQ ID NO. 21); β actin-ACCCACACTGTGCCCATCTACGAG (forward) (SEQ ID NO. 22), TCTCCTTAATGTCACGCACGATTTCC (reverse) (SEQ ID NO. 23). 18S rRNA Pre-developed TagMan Assays by Applied Biosystems was used as reference gene.
[0175]Patients and tumor specimens. The original patient material consisted of freshly frozen breast tumors from a population-based cohort of 315 women representing 65% of all breast cancers resected in Uppsala County from Jan. 1, 1987 to Dec. 31, 1989 (34, 35). The follow-up RNA expression profiling study was approved by the ethical committee at the Karolinska Institute, Stockholm, Sweden. Affymetrix (Santa Clara, Calif., USA) oligonucleotide microarrays (U133A8d3) were used to determine gene expression profiles in breast tumor samples. After exclusions based on RNA integrity and array quality control, expression profiles of 260 tumors were deemed suitable for further analysis. We selected the 69 ER-positive tumors from patients who underwent adjuvant tamoxifen therapy to assess the role of ERβ-regulated cell cycle and DNA replication genes in breast tumor biology. Clinico-pathological characteristics were derived from the patient records and from routine clinical measurements at the time of diagnosis as described elsewhere (34). Histopathological re-examination and grading according to Elston-Ellis was performed by an experienced breast cancer pathologist without prior knowledge of selected therapies and outcomes.
Results
ERβ Expression Elicited Similar Responses as Estrogen Treatment
[0176]ERβ expression was induced in the T47 Dbeta subline by tetracycline (TET) withdrawal (see Materials and Methods), and global gene expression profiles in the presence and absence of hormone were determined by human oligonucleotide microarrays (˜19,000 genes, Compugen, Jamesburg, N.J., USA) at 1, 6, 12, 16, and 30 h following induction. Responsive genes were defined as those that exhibited at least a 1.15 fold differential expression between untreated (-E2)-uninduced (+TET) controls and any induced and treated samples in the same direction for three consecutive time points or in four out of the five time points. 3509 genes or 19% (3509/18912) of the genes represented on the arrays were found to be responsive under at least one of the treatment conditions (+E2, +TET; -E2, -TET; or +E2, -TET) as compared to the untreated-uninduced controls. 47% (1662/3509) of the responsive genes were down-regulated as compared to the controls, whereas 53% (1847/3509) were up-regulated. Induction of the ERβ transcript was confirmed as an internal positive control.
[0177]We then examined the expression profiles of responsive genes by hierarchically clustering and visualizing the expression ratios using the Eisen Cluster and TreeView software (33) (see FIG. 1). Surprisingly, the global expression profiles under the three treatment conditions appeared nearly identical for up- (see FIG. 1A) and down-regulated (see FIG. 1B) genes. We assessed the overall similarities between the expression profiles of all genes on the arrays under each of the experimental conditions (uninduced-untreated control, E2 only, ERβ induction only, and E2+ERβ induction) and found a statistically significantly greater correlation (p<1×10-4 by Monte Carlo simulations) between the three test groups (E2 only, -ERβ induction only, and E2+ERβ induction) as compared to the control samples. These observations suggest that ERβ over-expression in the presence of ERα mimics the activation of ERα by estradiol. Earlier in vitro experiments demonstrated that ERα and ERβ can interact with the same ERE either as homodimers or heterodimers although interaction as heterodimers have not been formally determined (9-11). Our results extend these findings and indicate that one function of ERβ is to augment the basal transcriptional activity of ERα, even in the absence of ligand. These findings are also consistent with previous observations in osteosarcoma cells where ERα and ERβ share the majority of their estrogen responsive genes (31).
E2 Augments the Disruptive Effects of ERβ Over-Expression on Erα Activity
[0178]The significant similarity between E2 and ERβ transcriptional response appears to contradict the confirmed observation that ERβ blocks E2-induced cell proliferation in ERα poSitive cells (32). However, on detailed analysis of the expression profiles, two small clusters of genes emerged which may explain ERβ's antagonistic activity on cell growth. The first cluster, derived from FIG. 1A, contains genes whose expression levels were down-regulated following ERβ expression alone or E2 treatment and ERβ expression but up-regulated following E2 treatment alone (see FIG. 1A, magenta box). Among the genes found within this cluster, proliferating cell nuclear antigen (PCNA) and cell division cycle 6 (CDC6) are both known to be involved in DNA replication and have previously been shown to be estrogen responsive genes (29). The second cluster includes cell division cycle 2 (CDC2) and CDC28 protein kinase regulatory subunit 2 (CKS2), two cell cycle regulatory genes which were also down-regulated by ERβ expression in the presence and absence of E2. The highly specific contrary regulation of a small number of genes, specifically the down-regulation of genes involved in cell cycle regulation and DNA replication, present a plausible mechanism by which ERβ can block E2 induced cell, proliferation. That these genes were down-regulated even in the absence of E2 treatment in serum and estrogen-starved cells indicates that these transcriptional events are causative mechanisms rather than a reflection of cell proliferation inhibition by ERβ. These data support the notion that though the overall expression profile of ERβ/ERα expressing cells resembles that of ligand activated ERα, ERβ specifically alters the expression of a small number of key cell cycle associated genes that can explain its growth inhibitory effects.
[0179]E2 Augments the Disruptive Effects of ERβ Over-Expression on ERα Activity
[0180]In the absence of hormone, ERβ over-expression appeared to mimic E2 treatment in T-47D cells. The discrepancies that were observed, however, suggest that ERβ can indeed have targeted antagonistic effects on ERα function, specifically on the expression of genes promoting cell proliferation. We extended this analysis to examine the detailed effects of ERβ expression in the presence of the E2 ligand, by directly comparing gene expression levels between the two hormone treated sample groups: induced ERβ expression (-TET)+E2 vs. +E2 alone (+TET). Again, differentially expressed genes were defined as those that have a 1.15 fold change (E2+ERβ/E2) in the same direction for three consecutive time points or four out of the five time points. We posited that the addition of the ligand for ERα and ERβ might augment the differences between the two activated receptors.
[0181]As expected, in contrast to the previous results, we found that the addition of E2 to cells coexpressing ERβ and ERα more obviously perturbed the E2+ERα expression cassette in T-47D cells (N=1617 genes with 893 genes upregulated as compared 729 genes downregulated). From this expression data, we distinguished three models of interaction between ERα and ERβ when liganded with E2: synergistic, attenuation, and antagonistic models (see FIG. 2).
[0182]In the synergistic model, ERβ augments the ERα response to E2 (FIG. 2A). 18.7% (302/1617) of the differentially expressed genes exhibited greater responses when ERβ was expressed in the presence of E2 as compared to E2 treatment alone (changes in the same direction, but greater magnitude with ERβ see FIG. 2A). In 67.1% (1085/1617) of the genes, expression of ERβ resulted in the attenuation (changes in the same direction, but decrease in magnitude with ERβ) of the transcriptional response (see FIG. 2B). ERβ expression antagonized (changes in opposite directions) E2 response in 14.2% (230/1617) of differentially expressed genes (see FIG. 2C).
ERβ Inhibits the Cell Proliferative Effects of Estrogen and Erα by Specifically Blocking the Expression of Hormone Responsive Cell Cycle and DNA Replication Genes
[0183]We assessed in an unbiased fashion, whether the genes that were perturbed could be assigned to functional categories by gene ontology and whether genes of a specific function in cell proliferation were statistically significantly enriched when compared to the frequency of these genes represented on the microarray (gene ontology annotated by Compugen, Jamesburg, N.J., USA; see FIG. 2 and summarized in Table 2).
TABLE-US-00003 TABLE 2 Statistics of key gene ontology categories Gene Function % Total X2 p-Value E2 + ERβ UP-REGULATED Apoptosis 1.1% (10/893) 0.18590 Cell Cycle 1.5% (13/893) 0.82478 Cell Proliferation 0.4% (4/893) 0.41984 DNA Replication 0.4% (4/893) 0.80576 E2 + ERβ DOWN-REGULATED Apoptosis 0.4% (3/728) 0.31923 Cell Cycle 2.6% (19/728) 0.02471 Cell Proliferation 0.3% (2/728) 0.91690 DNA Replication 1.4% (10/728) 0.00175 ALL GENES ON ARRAY Apoptosis 0.7% (138/18912) -- Cell Cycle 1.5% (293/18912) -- Cell Proliferation 0.3% (56/18912) -- DNA Replication 0.5% (96/18912) --
We first observed that ERβ in the presence of E2 did not significantly up-regulate any specific functional class of genes. However, in the down-regulated cassette, we observed 1.7-fold (2.6% vs. 1.5%) enrichment in the frequency of cell cycle genes (chi-square p=0.0241) and a 2.8-fold (1.4% vs. 0.5%) enrichment in DNA replication genes (chi-square p=0.00175). When we divided the 728 down-regulated genes further into the three different models of interaction described in FIG. 2, we found that the cell cycle genes were overrepresented (5.6-fold; chi-square p=0.00077) in the group described by the antagonistic model; i.e., ERβ reversed the induction of cell cycle genes caused by the E2 ligand on ERα. DNA replication genes were enriched 3.2-fold (chi-square p=0.0008) in the group that showed attenuation by ERβ of the E2 mediated transcriptional induction (i.e., the attenuation model, see Table 4. Consistent with the earlier analysis, no functional gene group surveyed was augmented by ERβ. Thus, it appears that ERβ can specifically regulate the proliferative effects of E2 and ERα by reducing the expression levels of components of the cell cycle and DNA replication machinery.
TABLE-US-00004 TABLE 3 Statistics of key gene ontology categories by mechanism of regulation in 728 E2 + ERβ down-regulated genes Gene Function % Total X2 p-Value "SYNERGISTIC" Apoptosis 0% (0/129) 0.33018 Cell Cycle 0.8% (1/129) 0.47730 Cell Proliferation 0% (0/129) 0.53595 DNA Replication 0.8% (1/129) 0.67051 "ATTENUATION" Apoptosis 0.4% (2/492) 0.40303 Cell Cycle 1.8% (9/492) 0.11725 Cell Proliferation 0.2% (1/492) 0.70713 DNA Replication 1.6% (8/492) 0.00080 "ANTAGONISTIC" Apoptosis 0.9% (1/107) 0.80404 Cell Cycle 8.4% (6/107) 0.00077 Cell Proliferation 0.9% (1/107) 0.22828 DNA Replication 0.9% (1/107) 0.53640 ALL GENES ON ARRAY Apoptosis 0.7% (138/18912) -- Cell Cycle 1.5% (293/18912) -- Cell Proliferation 0.3% (56/18912) -- DNA Replication 0.5% (96/18912) --
Expression Profiles of ERβ Antagonized Cell Cycle Genes are Associated with ERβ Transcript Levels and Disease Outcome in ER+ Breast Cancer Patients
[0184]We sought to address whether this in vitro effect of ERβ on proliferation could explain some of the heterogeneity in biological behavior of ERα-positive breast cancer. We posited that higher ERβ levels would be correlated with lower expression of these specific DNA replication and cell cycle genes and, in turn, would be associated with better clinical outcome. To test this hypothesis, we examined ERβ transcript levels in primary breast cancers with the expression profiles of EAβ-regulated cell cycle genes identified in our in vitro analysis, and their association with clinical outcome.
[0185]To mimic our in vitro system, we specifically examined microarray expression data from 69 ERα-positive breast tumors from patients belonging to a previously described cohort (34, 35) from Uppsala, Sweden, who had received adjuvant tamoxifen therapy following surgery (see Materials and Methods). We extracted the expression data of the 6 cell cycle and 8 DNA replication genes (see Table 3) which were down-regulated by ERβ in the presence of E2 and classified the tumors base on their expression profiles. Two of the eight DNA replication genes were not found on the arrays used for the tumor studies so only 12 genes in total were available for analysis. We first assessed whether ERβ expression was associated with a decrease in the expression of the ERβ regulated cell cycle genes (i.e. the ERβ cassette). When examined, we found that for the majority of cassette genes (10/12; 83%) there is negative correlation between ERβ transcript levels and the ERβ transcription cassette (see Table 4). This is consistent with our in vitro findings that over-expression of ERβ leads to the down-regulation of the genes in the expression cassette. Four genes in the cassette, CDC2, DNA2L, CDC6, and CKS2, were significantly (student t-test P<0.05) associated with ERβ levels. The estrogen-dependent up-regulation of these genes and the suppression of their expression by ERβ over-expression were further validated by semi-quantitative PCR (see FIG. 3).
TABLE-US-00005 TABLE 4 Statistics of association with ERβ expression in 69 breast tumors. UniGene ID* Symbol Correlation p-value Hs.334562 CDC2 -0.30798 0.00151 Hs.194665 DNA2L -0.22919 0.01522 Hs.405958 CDC6 -0.16138 0.02774 Hs.83758 CKS2 -0.21564 0.04152 Hs.156346 TOP2A -0.11536 0.07061 Hs.436806 RAMP -0.20123 0.07442 Hs.436102 FLJ12484 0.05731 0.08202 Hs.115474 RFC3 -0.19135 0.11683 Hs.139226 RFC2 -0.09613 0.12292 Hs.135665 GAS2 0.05865 0.24376 Hs.142179 INCENP -0.03967 0.29616 Hs.234896 GMNN -0.07458 0.36950 Hs.22116 CDC14B** n/a n/a n/a PRO0245** n/a n/a *UniGene Build 175 **Not found on the arrays
We then used the four ERβ-regulated genes (CDC2, CKS2, CDC6, and DNA2L) with significant correlation with ERβ expression in the tumors as the functional ERβ cassette for subsequent clustering and associations with clinical parameters (see FIG. 4).
[0186]Using the expression of the ERβ cassette, the tumor samples were clustered into two groups with statistically significant association with 10-year relapse (chi-square P=0.00094) and death (chi-square P=0.03419), and tumor grade (chi-square P=0.00934).
[0187]To ascertain the correlation between the molecular signature and disease outcome, we compared the two patient clusters using disease free survival, DFS, (FIG. 4B) and disease specific survival, DSS, (FIG. 4C) as clinical endpoints. Patients with relatively higher levels of ERβ and lower expression of the cassette genes (FIG. 4A, Cluster 1; FIGS. 4B and 4C, black curves) have significantly improved outcomes than the group with lower levels of ERβ and higher levels of the expression cassette (FIG. 4A, Cluster 2; FIG. 4B and 4C, red curves) for both end points, DFS (p=0.0017) and DSS (p=0.0268). As predicted, we did not observe any significant association of the ERβ cassette expression profiles with disease outcome in 37 ERα-negative tumors from the same patient population data not shown). These results Were further validated in an independent dataset (36) of 45 ERα-positive tumors from patients who also underwent endocrine therapy. In this independent dataset of British patients; the ERβ expression cassette profiles also identified high and low ERβ expression cassette patient groups (see FIG. 5A) and to poor and good outcome groups for DFS (FIG. 5B, likelihood ratio P=0.0162) and DSS (FIG. 5C, likelihood ratio P=0.0192) respectively. Our findings indicate that the observed in vitro activity of ERβ on downstream target genes is directly recapitulated in primary breast tumors and has utility in further demarcating prognostic and therapeutic subgroups in the clinical Setting. In addition, these results implicate the selective ERβ transcriptional effects as identified in vitro in defining clinical behavior of human breast cancers.
Discussion
[0188]Original characterization of ERβ function indicated its ability to form homodimers and heterodimers (with ERα) and to bind the same consensus ERE sequences in electrophoretic mobility shift experiments (9). However, ERβ biological effects are considerably different from that of ERα: ERβ expression alone, unlike hormone treatment, was not sufficient to drive cell proliferation (32). In cell lines, over expression of ERβ inhibited E2-driven and ERα-mediated cell proliferation (32). Our finding that over-expression of ERβ in ERα+/ERβ- T-47D cells elicited similar gene expression profiles as hormone treated ERα positive cells was, therefore, surprising. These results suggested that ERα/β heterodimers or ERβ homodimers can increase the basal activity of the ER complex. However, a more detailed genome-wide assessment of transcriptional activity provide strong evidence that over-expression of ERβ in T-47D cells does not fully Mimic E2 treatment, and that there are levels of ordered functional complexity to this interaction: despite the similarity of expression control, a small subset of genes, such as CDC2, CDC6, and PCNA (see Table 2, part 2 and FIG. 3), previously shown to be up-regulated by E2 (29), were down-regulated by ERβ expression. Therefore, in spite of the overall similarities in the ligand-independent functions of ERβ in the presence of ERα and the hormone induced activity of ERα, there are specific regulatory pathways targeted by ERβ that may antagonize key ERα functions (e.g. proliferation).
[0189]The initial phenotypic characterization of the cell line showed that over-expression of ERβ blocked E2-driven and ERα-mediated cell proliferation (32). When we directly compared gene expression profiles following ERβ over-expression and hormone treatment to those obtained from cells subjected to hormone treatment only, we now observe the up-regulation of 829 genes and down-regulation of 729 genes, suggesting that ERβ can significantly alter the ERα-mediated transcriptional program mainly in the presence of estrogens. Taken together, our genome-wide analysis has uncovered differential effects of ERβ in a ligand-dependent context: in the absence of ligand, ERβ largely mimics ERα effects except in a small number of genes that affect cell cycle and DNA replication, in the presence of the E2 ligand, ERβ has more apparent antagonistic effects on ERα function. Indeed, statistical analysis indicated a significant enrichment of genes from these two functional categories within the population of genes down-regulated by ERβ in the presence of E2 (see Tables 3 and 4).
[0190]One of the difficulties in assessing the biological significance of such associations between in vitro gene expression changes and the cellular response is that a number of genes coordinately expressed as a cassette (e.g., the ERβ cassette) are involved. One-by-one targeting of the genes in the cassette is not only experimentally problematic but also raises the question of relevance given that an entire gene set may need to be altered. We pursued an alternative approach to determining the in vivo relevance of these findings in the engineered cell line by exploring the association of ERβ transcript levels and the key ERβ suppressed cell cycle and DNA replication genes, in disease outcome of breast cancer patients. We hypothesized that if our in vitro findings were biologically important, then its configuration should be captured in clinical tumors and its biological impact should recapitulate that found in cell culture.
[0191]To this end, we first found significant association of the expression profiles of four genes, CDC2, CKS2, DNA2L, and CDC6, with ERβ transcript levels in the breast tumors, consistent with the inverse correlation (high ERβ; low cell cycle/DNA replication genes) observed in the cell line model. CDC2 is a cyclin-dependent kinase which regulates G1-S and G2-M phases of the cell cycle; CKS2 encodes the essential regulatory subunit of cyclin-dependent kinases; and CDC6 and DNA2L function in the pre-replication complex formation and unwinding of replicating DNA respectively. Tumor samples were then hierarchically clustered into two distinct groups, using the expression profiles of this four-gene expression cassette and ERβ (see FIG. 4). We found significant association of the patient groups with tumor grade (which we consider a clinical surrogate for cell cycle and DNA replication), and with 10-year relapse and death. Moreover, the patient cluster with high ERβ and low ERβ downstream gene expression have significantly higher probability of disease-free and disease specific survival over time as compared to those with the opposite profiles. These results suggest that the ERβ regulated genes identified in our in vitro studies are also involved in the biology of primary malignant breast tumors and reveal protective effects of ERβ in disease prognosis. That this effect is limited to ERα-positive subgroup is also consistent with our observations that the main ERβ effect is in the presence of ERα and the E2 ligand. Previously, we had identified that ER positive tumors exhibited significant differences detected by expression profiling associated with variances in overall survival (36). Using the same dataset, we assessed the impact of ERβ and its unique downstream expression cassette and found that this minimal gene set alone could account for the majority of the survival differences. This suggests that ERβ status is the major driver for clinical heterogeneity in ER positive tumors.
[0192]Our findings are consistent with a recent report by Hopp and colleagues where they found that loW ERβ protein levels are associated with poor disease-free and over-all survival in ER-positive patients treated with adjuvant tamoxifen therapy (37). In contrast, another group reported the lack of association between ERβ transcript-levels and disease outcome following endocrine therapy in a prospective study (38). One possible explanation for the discrepancy between the prospective study and the previous protein level study and the expression cassette data reported here is that protein levels and expression profiles of downstream ERβ regulated genes are a more proximal measure of ERβ activity than the transcript levels alone.
[0193]A reasonable question is whether the ERβ downstream genes harbor the transcriptional control elements associated with the estrogen receptors, i.e., EREs. Our assessment of the 5' regulatory regions of the four key downstream genes CDC2, CKS2, DNA2L, and CDC6 showed no evidence for an acceptable ERE (data not shown). This raises the possibility that either the expression of these genes involve trans-elements such as other transcription factors induced by ERβ, or that ERβ is acting as a co-factor for other transcription factors such as AP-1 (39, 40).
[0194]Taken together, our observations also suggest that molecules directly targeting ERα/ERβ interactions or ERβ mimics could be a novel strategy to develop estrogen response modifiers for the management of ERα-positive breast cancers.
[0195]It is to be understood that the above description in intended to be, illustrative and not restrictive. Many variations of the invention will be apparent to those of skill in the art upon reviewing the above description. The scope of the invention should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. All cited references, including patent and non-patent literature, are incorporated herewith by reference in their entireties for all purposes.
REFERENCES
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Sequence CWU
1
2311235DNAHomo sapiens 1gggggggggg ggcacttggc ttcaaagctg gctcttggaa
attgagcgga gagcgacgcg 60gttgttgtag ctgccgctgc ggccgccgcg gaataataag
ccgggatcta ccatacccat 120tgactaacta tggaagatta taccaaaata gagaaaattg
gagaaggtac ctatggagtt 180gtgtataagg gtagacacaa aactacaggt caagtggtag
ccatgaaaaa aatcagacta 240gaaagtgaag aggaaggggt tcctagtact gcaattcggg
aaatttctct attaaaggaa 300cttcgtcatc caaatatagt cagtcttcag gatgtgctta
tgcaggattc caggttatat 360ctcatctttg agtttctttc catggatctg aagaaatact
tggattctat ccctcctggt 420cagtacatgg attcttcact tgttaagagt tatttatacc
aaatcctaca ggggattgtg 480ttttgtcact ctagaagagt tcttcacaga gacttaaaac
ctcaaaatct cttgattgat 540gacaaaggaa caattaaact ggctgatttt ggccttgcca
gagcttttgg aatacctatc 600agagtatata cacatgaggt agtaacactc tggtacagat
ctccagaagt attgctgggg 660tcagctcgtt actcaactcc agttgacatt tggagtatag
gcaccatatt tgctgaacta 720gcaactaaga aaccactttt ccatggggat tcagaaattg
atcaactctt caggattttc 780agagctttgg gcactcccaa taatgaagtg tggccagaag
tggaatcttt acaggactat 840aagaatacat ttcccaaatg gaaaccagga agcctagcat
cccatgtcaa aaacttggat 900gaaaatggct tggatttgct ctcgaaaatg ttaatctatg
atccagccaa acgaatttct 960ggcaaaatgg cactgaatca tccatatttt aatgatttgg
acaatcagat taagaagatg 1020tagctttctg acaaaaagtt tccatatgtt atgtcaacag
atagttgtgt ttttattgtt 1080aactcttgtc tatttttgtc ttatatatat ttctttgtta
tcaaacttca gctgtacttc 1140gtcttctaat ttcaaaaata taacttaaaa atgtaaatat
tctatatgaa tttaaatata 1200attctgtaaa tgtgaaaaaa aaaaaaaaaa aaaaa
12352297PRTHomo sapiens 2Met Glu Asp Tyr Thr Lys
Ile Glu Lys Ile Gly Glu Gly Thr Tyr Gly1 5
10 15Val Val Tyr Lys Gly Arg His Lys Thr Thr Gly Gln
Val Val Ala Met 20 25 30Lys
Lys Ile Arg Leu Glu Ser Glu Glu Glu Gly Val Pro Ser Thr Ala 35
40 45Ile Arg Glu Ile Ser Leu Leu Lys Glu
Leu Arg His Pro Asn Ile Val 50 55
60Ser Leu Gln Asp Val Leu Met Gln Asp Ser Arg Leu Tyr Leu Ile Phe65
70 75 80Glu Phe Leu Ser Met
Asp Leu Lys Lys Tyr Leu Asp Ser Ile Pro Pro 85
90 95Gly Gln Tyr Met Asp Ser Ser Leu Val Lys Ser
Tyr Leu Tyr Gln Ile 100 105
110Leu Gln Gly Ile Val Phe Cys His Ser Arg Arg Val Leu His Arg Asp
115 120 125Leu Lys Pro Gln Asn Leu Leu
Ile Asp Asp Lys Gly Thr Ile Lys Leu 130 135
140Ala Asp Phe Gly Leu Ala Arg Ala Phe Gly Ile Pro Ile Arg Val
Tyr145 150 155 160Thr His
Glu Val Val Thr Leu Trp Tyr Arg Ser Pro Glu Val Leu Leu
165 170 175Gly Ser Ala Arg Tyr Ser Thr
Pro Val Asp Ile Trp Ser Ile Gly Thr 180 185
190Ile Phe Ala Glu Leu Ala Thr Lys Lys Pro Leu Phe His Gly
Asp Ser 195 200 205Glu Ile Asp Gln
Leu Phe Arg Ile Phe Arg Ala Leu Gly Thr Pro Asn 210
215 220Asn Glu Val Trp Pro Glu Val Glu Ser Leu Gln Asp
Tyr Lys Asn Thr225 230 235
240Phe Pro Lys Trp Lys Pro Gly Ser Leu Ala Ser His Val Lys Asn Leu
245 250 255Asp Glu Asn Gly Leu
Asp Leu Leu Ser Lys Met Leu Ile Tyr Asp Pro 260
265 270Ala Lys Arg Ile Ser Gly Lys Met Ala Leu Asn His
Pro Tyr Phe Asn 275 280 285Asp Leu
Asp Asn Gln Ile Lys Lys Met 290 2953948DNAHomo sapiens
3ccattgacta actatggaag attataccaa aatagagaaa attggagaag gtacctatgg
60agttgtgtat aagggtagac acaaaactac aggtcaagtg gtagccatga aaaaaatcag
120actagaaagt gaagaggaag gggttcctag tactgcaatt cgggaaattt ctctattaaa
180ggaacttcgt catccaaata tagtcagtct tcaggatgtg cttatgcagg attccaggtt
240atatctcatc tttgagtttc tttccatgga tctgaagaaa tacttggatt ctatccctcc
300tggtcagtac atggattctt cacttgttaa ggtagtaaca ctctggtaca gatctccaga
360agtattgctg gggtcagctc gttactcaac tccagttgac atttggagta taggcaccat
420atttgctgaa ctagcaacta agaaaccact tttccatggg gattcagaaa ttgatcaact
480cttcaggatt ttcagagctt tgggcactcc caataatgaa gtgtggccag aagtggaatc
540tttacaggac tataagaata catttcccaa atggaaacca ggaagcctag catcccatgt
600caaaaacttg gatgaaaatg gcttggattt gctctcgaaa atgttaatct atgatccagc
660caaacgaatt tctggcaaaa tggcactgaa tcatccatat tttaatgatt tggacaatca
720gattaagaag atgtagcttt ctgacaaaaa gtttccatat gttatgtcaa cagatagttg
780tgtttttatt gttaactctt gtctattttt gtcttatata tatttctttg ttatcaaact
840tcagctgtac ttcgtcttct aatttcaaaa atataactta aaaatgtaaa tattctatat
900gaatttaaat ataattctgt aaatgtgaaa aaaaaaaaaa aaaaaaaa
9484240PRTHomo sapiens 4Met Glu Asp Tyr Thr Lys Ile Glu Lys Ile Gly Glu
Gly Thr Tyr Gly1 5 10
15Val Val Tyr Lys Gly Arg His Lys Thr Thr Gly Gln Val Val Ala Met
20 25 30Lys Lys Ile Arg Leu Glu Ser
Glu Glu Glu Gly Val Pro Ser Thr Ala 35 40
45Ile Arg Glu Ile Ser Leu Leu Lys Glu Leu Arg His Pro Asn Ile
Val 50 55 60Ser Leu Gln Asp Val Leu
Met Gln Asp Ser Arg Leu Tyr Leu Ile Phe65 70
75 80Glu Phe Leu Ser Met Asp Leu Lys Lys Tyr Leu
Asp Ser Ile Pro Pro 85 90
95Gly Gln Tyr Met Asp Ser Ser Leu Val Lys Val Val Thr Leu Trp Tyr
100 105 110Arg Ser Pro Glu Val Leu
Leu Gly Ser Ala Arg Tyr Ser Thr Pro Val 115 120
125Asp Ile Trp Ser Ile Gly Thr Ile Phe Ala Glu Leu Ala Thr
Lys Lys 130 135 140Pro Leu Phe His Gly
Asp Ser Glu Ile Asp Gln Leu Phe Arg Ile Phe145 150
155 160Arg Ala Leu Gly Thr Pro Asn Asn Glu Val
Trp Pro Glu Val Glu Ser 165 170
175Leu Gln Asp Tyr Lys Asn Thr Phe Pro Lys Trp Lys Pro Gly Ser Leu
180 185 190Ala Ser His Val Lys
Asn Leu Asp Glu Asn Gly Leu Asp Leu Leu Ser 195
200 205Lys Met Leu Ile Tyr Asp Pro Ala Lys Arg Ile Ser
Gly Lys Met Ala 210 215 220Leu Asn His
Pro Tyr Phe Asn Asp Leu Asp Asn Gln Ile Lys Lys Met225
230 235 24053053DNAHomo sapiens
5gagcgcggct ggagtttgct gctgccgctg tgcagtttgt tcaggggctt gtggtggtga
60gtccgagagg ctgcgtgtga gagacgtgag aaggatcctg cactgaggag gtggaaagaa
120gaggattgct cgaggaggcc tggggtctgt gaggcagcgg agctgggtga aggctgcggg
180ttccggcgag gcctgagctg tgctgtcgtc atgcctcaaa cccgatccca ggcacaggct
240acaatcagtt ttccaaaaag gaagctgtct cgggcattga acaaagctaa aaactccagt
300gatgccaaac tagaaccaac aaatgtccaa accgtaacct gttctcctcg tgtaaaagcc
360ctgcctctca gccccaggaa acgtctgggc gatgacaacc tatgcaacac tccccattta
420cctccttgtt ctccaccaaa gcaaggcaag aaagagaatg gtccccctca ctcacataca
480cttaagggac gaagattggt atttgacaat cagctgacaa ttaagtctcc tagcaaaaga
540gaactagcca aagttcacca aaacaaaata ctttcttcag ttagaaaaag tcaagagatc
600acaacaaatt ctgagcagag atgtccactg aagaaagaat ctgcatgtgt gagactattc
660aagcaagaag gcacttgcta ccagcaagca aagctggtcc tgaacacagc tgtcccagat
720cggctgcctg ccagggaaag ggagatggat gtcatcagga atttcttgag ggaacacatc
780tgtgggaaaa aagctggaag cctttacctt tctggtgctc ctggaactgg aaaaactgcc
840tgcttaagcc ggattctgca agacctcaag aaggaactga aaggctttaa aactatcatg
900ctgaattgca tgtccttgag gactgcccag gctgtattcc cagctattgc tcaggagatt
960tgtcaggaag aggtatccag gccagctggg aaggacatga tgaggaaatt ggaaaaacat
1020atgactgcag agaagggccc catgattgtg ttggtattgg acgagatgga tcaactggac
1080agcaaaggcc aggatgtatt gtacacgcta tttgaatggc catggctaag caattctcac
1140ttggtgctga ttggtattgc taataccctg gatctcacag atagaattct acctaggctt
1200caagctagag aaaaatgtaa gccacagctg ttgaacttcc caccttatac cagaaatcag
1260atagtcacta ttttgcaaga tcgacttaat caggtatcta gagatcaggt tctggacaat
1320gctgcagttc aattctgtgc ccgcaaagtc tctgctgttt caggagatgt tcgcaaagca
1380ctggatgttt gcaggagagc tattgaaatt gtagagtcag atgtcaaaag ccagactatt
1440ctcaaaccac tgtctgaatg taaatcacct tctgagcctc tgattcccaa gagggttggt
1500cttattcaca tatcccaagt catctcagaa gttgatggta acaggatgac cttgagccaa
1560gaaggagcac aagattcctt ccctcttcag cagaagatct tggtttgctc tttgatgctc
1620ttgatcaggc agttgaaaat caaagaggtc actctgggga agttatatga agcctacagt
1680aaagtctgtc gcaaacagca ggtggcggct gtggaccagt cagagtgttt gtcactttca
1740gggctcttgg aagccagggg cattttagga ttaaagagaa acaaggaaac ccgtttgaca
1800aaggtgtttt tcaagattga agagaaagaa atagaacatg ctctgaaaga taaagcttta
1860attggaaata tcttagctac tggattgcct taaattcttc tcttacaccc cacccgaaag
1920tattcagctg gcatttagag agctacagtc ttcattttag tgctttacac attcgggcct
1980gaaaacaaat atgacctttt ttacttgaag ccaatgaatt ttaatctata gattctttaa
2040tattagcaca gaataatatc tttgggtctt actattttta cccataaaag tgaccaggta
2100gacccttttt aattacattc actacttcta ccacttgtgt atctctagcc aatgtgcttg
2160caagtgtaca gatctgtgta gaggaatgtg tgtatattta cctcttcgtt tgctcaaaca
2220tgagtgggta tttttttgtt tgtttttttt gttgttgttg tttttgaggc gcgtctcacc
2280ctgttgccca ggctggagtg caatggcgcg ttctctgctc actacagcac ccgcttccca
2340ggttgaagtg attctcttgc ctcagcctcc cgagtagctg ggattacagg tgcccaccac
2400cgcgcccagc taatttttta atttttagta gagacagggt tttaccatgt tggccaggct
2460ggtcttgaac tcctgaccct caagtgatct gcccaccttg gcctccctaa gtgctgggat
2520tataggcgtg agccaccatg ctcagccatt aaggtatttt gttaagaact ttaagtttag
2580ggtaagaaga atgaaaatga tccagaaaaa tgcaagcaag tccacatgga gatttggagg
2640acactggtta aagaatttat ttctttgtat agtatactat gttcatggtg cagatactac
2700aacattgtgg cattttagac tcgttgagtt tcttgggcac tcccaagggc gttggggtca
2760taaggagact ataactctac agattgtgaa tatatttatt ttcaagttgc attctttgtc
2820tttttaagca atcagatttc aagagagctc aagctttcag aagtcaatgt gaaaattcct
2880tcctaggctg tcccacagtc tttgctgccc ttagatgaag ccacttgttt caagatgact
2940actttggggt tgggttttca tctaaacaca tttttccagt cttattagat aaattagtcc
3000atatggttgg ttaatcaaga gccttctggg tttggtttgg tggcattaaa tgg
30536560PRTHomo sapiens 6Met Pro Gln Thr Arg Ser Gln Ala Gln Ala Thr Ile
Ser Phe Pro Lys1 5 10
15Arg Lys Leu Ser Arg Ala Leu Asn Lys Ala Lys Asn Ser Ser Asp Ala
20 25 30Lys Leu Glu Pro Thr Asn Val
Gln Thr Val Thr Cys Ser Pro Arg Val 35 40
45Lys Ala Leu Pro Leu Ser Pro Arg Lys Arg Leu Gly Asp Asp Asn
Leu 50 55 60Cys Asn Thr Pro His Leu
Pro Pro Cys Ser Pro Pro Lys Gln Gly Lys65 70
75 80Lys Glu Asn Gly Pro Pro His Ser His Thr Leu
Lys Gly Arg Arg Leu 85 90
95Val Phe Asp Asn Gln Leu Thr Ile Lys Ser Pro Ser Lys Arg Glu Leu
100 105 110Ala Lys Val His Gln Asn
Lys Ile Leu Ser Ser Val Arg Lys Ser Gln 115 120
125Glu Ile Thr Thr Asn Ser Glu Gln Arg Cys Pro Leu Lys Lys
Glu Ser 130 135 140Ala Cys Val Arg Leu
Phe Lys Gln Glu Gly Thr Cys Tyr Gln Gln Ala145 150
155 160Lys Leu Val Leu Asn Thr Ala Val Pro Asp
Arg Leu Pro Ala Arg Glu 165 170
175Arg Glu Met Asp Val Ile Arg Asn Phe Leu Arg Glu His Ile Cys Gly
180 185 190Lys Lys Ala Gly Ser
Leu Tyr Leu Ser Gly Ala Pro Gly Thr Gly Lys 195
200 205Thr Ala Cys Leu Ser Arg Ile Leu Gln Asp Leu Lys
Lys Glu Leu Lys 210 215 220Gly Phe Lys
Thr Ile Met Leu Asn Cys Met Ser Leu Arg Thr Ala Gln225
230 235 240Ala Val Phe Pro Ala Ile Ala
Gln Glu Ile Cys Gln Glu Glu Val Ser 245
250 255Arg Pro Ala Gly Lys Asp Met Met Arg Lys Leu Glu
Lys His Met Thr 260 265 270Ala
Glu Lys Gly Pro Met Ile Val Leu Val Leu Asp Glu Met Asp Gln 275
280 285Leu Asp Ser Lys Gly Gln Asp Val Leu
Tyr Thr Leu Phe Glu Trp Pro 290 295
300Trp Leu Ser Asn Ser His Leu Val Leu Ile Gly Ile Ala Asn Thr Leu305
310 315 320Asp Leu Thr Asp
Arg Ile Leu Pro Arg Leu Gln Ala Arg Glu Lys Cys 325
330 335Lys Pro Gln Leu Leu Asn Phe Pro Pro Tyr
Thr Arg Asn Gln Ile Val 340 345
350Thr Ile Leu Gln Asp Arg Leu Asn Gln Val Ser Arg Asp Gln Val Leu
355 360 365Asp Asn Ala Ala Val Gln Phe
Cys Ala Arg Lys Val Ser Ala Val Ser 370 375
380Gly Asp Val Arg Lys Ala Leu Asp Val Cys Arg Arg Ala Ile Glu
Ile385 390 395 400Val Glu
Ser Asp Val Lys Ser Gln Thr Ile Leu Lys Pro Leu Ser Glu
405 410 415Cys Lys Ser Pro Ser Glu Pro
Leu Ile Pro Lys Arg Val Gly Leu Ile 420 425
430His Ile Ser Gln Val Ile Ser Glu Val Asp Gly Asn Arg Met
Thr Leu 435 440 445Ser Gln Glu Gly
Ala Gln Asp Ser Phe Pro Leu Gln Gln Lys Ile Leu 450
455 460Val Cys Ser Leu Met Leu Leu Ile Arg Gln Leu Lys
Ile Lys Glu Val465 470 475
480Thr Leu Gly Lys Leu Tyr Glu Ala Tyr Ser Lys Val Cys Arg Lys Gln
485 490 495Gln Val Ala Ala Val
Asp Gln Ser Glu Cys Leu Ser Leu Ser Gly Leu 500
505 510Leu Glu Ala Arg Gly Ile Leu Gly Leu Lys Arg Asn
Lys Glu Thr Arg 515 520 525Leu Thr
Lys Val Phe Phe Lys Ile Glu Glu Lys Glu Ile Glu His Ala 530
535 540Leu Lys Asp Lys Ala Leu Ile Gly Asn Ile Leu
Ala Thr Gly Leu Pro545 550 555
5607627DNAHomo sapiens 7agtctccggc gagttgttgc ctgggctgga cgtggttttg
tctgctgcgc ccgctcttcg 60cgctctcgtt tcattttctg cagcgcgcca cgaggatggc
ccacaagcag atctactact 120cggacaagta cttcgacgaa cactacgagt accggcatgt
tatgttaccc agagaacttt 180ccaaacaagt acctaaaact catctgatgt ctgaagagga
gtggaggaga cttggtgtcc 240aacagagtct aggctgggtt cattacatga ttcatgagcc
agaaccacat attcttctct 300ttagacgacc tcttccaaaa gatcaacaaa aatgaagttt
atctggggat cgtcaaatct 360ttttcaaatt taatgtatat gtgtatataa ggtagtattc
agtgaatact tgagaaatgt 420acaaatcttt catccatacc tgtgcatgag ctgtattctt
cacagcaaca gagctcagtt 480aaatgcaact gcaagtaggt tactgtaaga tgtttaagat
aaaagttctt ccagtcagtt 540tttctcttaa gtgcctgttt gagtttactg aaacagttta
cttttgttca ataaagtttg 600tatgttgcat ttaaaaaaaa aaaaaaa
627879PRTHomo sapiens 8Met Ala His Lys Gln Ile Tyr
Tyr Ser Asp Lys Tyr Phe Asp Glu His1 5 10
15Tyr Glu Tyr Arg His Val Met Leu Pro Arg Glu Leu Ser
Lys Gln Val 20 25 30Pro Lys
Thr His Leu Met Ser Glu Glu Glu Trp Arg Arg Leu Gly Val 35
40 45Gln Gln Ser Leu Gly Trp Val His Tyr Met
Ile His Glu Pro Glu Pro 50 55 60His
Ile Leu Leu Phe Arg Arg Pro Leu Pro Lys Asp Gln Gln Lys65
70 7594415DNAHomo sapiens 9atgaagactc cctgtattcc
cagtcctaag caagggagca aaggcagggg acagagccct 60gctgctcagg tgaggggccc
cacgtggaac gcgccggcgc gggaggggcg gcctggcgca 120ggtcatttgg gacatctgcg
ggttggcgca tgcgcgcgag gtgcgcaggc tcgcgccttt 180ttcccttttc taagctttct
gtgttacccc cggttccgct gtcttttctg tctacagttt 240gcgatccccg cgtccaggat
ggagcagctg aacgaactgg agctgctgat ggagaagagt 300ttttgggagg aggcggagct
gccggcggag ctatttcaga agaaagtggt agcttccttt 360ccaagaacag ttctgagcac
aggaatggat aaccggtacc tggtgttggc agtcaatact 420gtacagaaca aagagggaaa
ctgtgaaaag cgcctggtca tcactgcttc acagtcacta 480gaaaataaag aactatgcat
ccttaggaat gactggtgtt ctgttccagt agagccagga 540gatatcattc atttggaggg
agactgcaca tctgacactt ggataataga taaagatttt 600ggatatttga ttctgtatcc
agacatgctg atttctggca ccagcatagc cagtagtatt 660cgatgtatga gaagagctgt
cctgagtgaa acttttagga gctctgatcc agccacacgc 720caaatgctaa ttggtacggt
tctccatgag gtgtttcaaa aagccataaa taatagcttt 780gccccagaaa agctacaaga
acttgctttt caaacaattc aagaaataag acatttgaag 840gaaatgtacc gcttaaatct
aagtcaagat gaaataaaac aagaagtaga ggactatctt 900ccttcgtttt gtaaatgggc
aggagatttc atgcataaaa acacttcgac tgacttccct 960cagatgcagc tctctctgcc
aagtgataat agtaaggata attcaacatg taacattgaa 1020gtcgtgaaac caatggatat
tgaagaaagc atttggtccc ctaggtttgg attgaaaggc 1080aaaatagatg ttacagttgg
tgtgaaaata catcgagggt ataaaacaaa atacaagata 1140atgccgctgg aacttaaaac
tggcaaagaa tcaaattcta ttgaacaccg tagtcaggtt 1200gttctgtaca ctctactaag
ccaagagaga agagctgatc cagaggctgg cttgcttctc 1260tacctcaaga ctggtcagat
gtaccctgtg cctgccaacc atctagataa aagagaatta 1320ttaaagctaa gaaaccagat
ggcattctca ttgtttcacc gtattagcaa atctgctact 1380agacagaaga cacagcttgc
ttctttgcca caaataattg aggaagagaa aacttgtaaa 1440tattgttcac aaattggcaa
ttgtgctctt tatagcagag cagttgaaca acagatggat 1500tgtagttcag tcccaattgt
gatgctgccc aaaatagaag aagaaaccca gcatctgaag 1560caaacacact tagaatattt
cagcctttgg tgtctaatgt taaccctgga gtcacaatcg 1620aaggataata aaaagaatca
ccaaaatatc tggctaatgc ctgcttcgga aatggagaag 1680agtggcagtt gcattggaaa
cctgattaga atggaacatg taaagatagt ttgtgatggg 1740caatatttac ataatttcca
atgtaaacat ggtgccatac ctgtcacaaa tctaatggca 1800ggtgacagag ttattgtaag
tggagaagaa aggtcactgt ttgctttgtc tagaggatat 1860gtgaaggaga ttaacatgac
aacagtaact tgtttattag acagaaactt gtcggtcctt 1920ccagaatcaa ctttgttcag
attagaccaa gaagaaaaaa attgtgatat agatacccca 1980ttaggaaatc tttccaaatt
gatggaaaac acgtttgtca gcaaaaaact tcgagattta 2040attattgact ttcgtgaacc
tcagtttata tcctacctta gttctgttct tccacatgat 2100gcaaaggata cagttgcctg
cattctaaag ggtttgaata agcctcagag gcaagcgatg 2160aaaaaggtac ttctttcaaa
agactacaca ctcatcgtgg gtatgcctgg gacaggaaaa 2220acaactacga tatgtactct
cgtaagaatt ctctacgcct gtggttttag cgttttgttg 2280accagctata cacactctgc
tgttgacaat attcttttga agttagccaa gtttaaaata 2340ggatttttgc gtttgggtca
gattcagaag gttcatccag ctatccagca atttacagag 2400caagaaattt gcagatcaaa
gtccattaaa tccttagctc ttctagaaga actctacaat 2460agtcaactta tagttgcaac
aacatgtatg ggaataaacc atccaatatt ttcccgtaaa 2520atttttgatt tttgtattgt
ggatgaagcc tctcaaatta gccaaccaat ttgtctgggc 2580cccctttttt tttcacggag
atttgtgtta gtgggggacc atcagcagct tcctcccctg 2640gtgctaaacc gtgaagcaag
agctcttggc atgagtgaaa gcttattcaa gaggctggag 2700cagaataaga gtgctgttgt
acagttaacc gtgcagtaca gaatgaacag taaaattatg 2760tccttaagta ataagctgac
ctatgagggc aagctggagt gtggatcaga caaagtggcc 2820aatgcagtga taaacctacg
tcactttaaa gatgtgaagc tggaactgga attttatgct 2880gactattctg ataatccttg
gttgatggga gtatttgaac ccaacaatcc tgtttgtttc 2940cttaatacag acaaggttcc
agcgccagaa caagttgaaa aaggtggtgt gagcaatgta 3000acagaagcca aactcatagt
tttcctaacc tccatttttg ttaaggctgg atgcagtccc 3060tctgatattg gtattattgc
accgtacagg cagcaattaa agatcatcaa tgatttattg 3120gcacgttcta ttgggatggt
cgaagttaat acagtagaca aataccaagg aagggacaaa 3180agtattgtcc tagtatcttt
tgttagaagt aataaggatg gaactgttgg tgaactcttg 3240aaagattggc gacgtcttaa
tgttgctata accagagcca aacataaact gattcttctg 3300gggtgtgtgc cctcactaaa
ttgctatcct cctttggaga agctgcttaa tcatttaaac 3360tcagaaaaat taatcattga
tcttccatca agagaacatg aaagtctttg ccacatattg 3420ggtgactttc aaagagaata
aaacactatt tcccttgcct tttcatacta gggcagtatc 3480tcctctagct agtgcccata
cagaaaattc tatcaccata caaaatttaa tgcagtattt 3540atgttttaaa gcacaggtgt
accgaaaact gtgaaaagtc tgaatttatg ggttctatgc 3600atgcattttt gcctaaccta
gagaaagagt ttgataaatt tttaccagct ttgaagatgg 3660attaactttt gactttgagc
tttaaacttt taagtcagac atttcaggac taatttgatt 3720ttgtagatat cattgtaaga
actttatttg aaagactgaa taaagggatt tgatttgttt 3780tcatcattta agcacagtct
tgtgatgatg agaacataag tgtgattctt ttctgtattt 3840tgaggtccct aatccaaagc
ccattttgct aggatttttt ctgctatcag atgtgttttc 3900actctaaacc tagtctttta
tgacatgaat tgattacttc ctgttaattt tctatcctcc 3960cttactatcc tccttttttg
ttttcagtat tcagtatttc agtattctag agtagatttt 4020gatataaaag aaaataattc
ttacatcatc ttttgcaaca aattttgttt tctgaattga 4080taataaattt aaaaagttga
ttcctatttt cacatatgtt catatgcccc tatgtttggg 4140ggtatcactc agttttccct
tttttgtgta aagatgtttt gtaaaacaaa attgtctcaa 4200agtgattata ttatatatat
aaaaagtaac agattttaac aaaggttaaa agattcttgg 4260ggtaacagat tcttctgggg
cttggaaatc ttccatttct cttgagggtt ttttttaatg 4320agtgttaaat atgttaaaat
ttttatttct acctcatgtg tttttttaaa ttattacttg 4380aagtttttta tttaataaat
tttttctact aatgg 4415101146PRTHomo sapiens
10Met Lys Thr Pro Cys Ile Pro Ser Pro Lys Gln Gly Ser Lys Gly Arg1
5 10 15Gly Gln Ser Pro Ala Ala
Gln Val Arg Gly Pro Thr Trp Asn Ala Pro 20 25
30Ala Arg Glu Gly Arg Pro Gly Ala Gly His Leu Gly His
Leu Arg Val 35 40 45Gly Ala Cys
Ala Arg Gly Ala Gln Ala Arg Ala Phe Phe Pro Phe Leu 50
55 60Ser Phe Leu Cys Tyr Pro Arg Phe Arg Cys Leu Phe
Cys Leu Gln Phe65 70 75
80Ala Ile Pro Ala Ser Arg Met Glu Gln Leu Asn Glu Leu Glu Leu Leu
85 90 95Met Glu Lys Ser Phe Trp
Glu Glu Ala Glu Leu Pro Ala Glu Leu Phe 100
105 110Gln Lys Lys Val Val Ala Ser Phe Pro Arg Thr Val
Leu Ser Thr Gly 115 120 125Met Asp
Asn Arg Tyr Leu Val Leu Ala Val Asn Thr Val Gln Asn Lys 130
135 140Glu Gly Asn Cys Glu Lys Arg Leu Val Ile Thr
Ala Ser Gln Ser Leu145 150 155
160Glu Asn Lys Glu Leu Cys Ile Leu Arg Asn Asp Trp Cys Ser Val Pro
165 170 175Val Glu Pro Gly
Asp Ile Ile His Leu Glu Gly Asp Cys Thr Ser Asp 180
185 190Thr Trp Ile Ile Asp Lys Asp Phe Gly Tyr Leu
Ile Leu Tyr Pro Asp 195 200 205Met
Leu Ile Ser Gly Thr Ser Ile Ala Ser Ser Ile Arg Cys Met Arg 210
215 220Arg Ala Val Leu Ser Glu Thr Phe Arg Ser
Ser Asp Pro Ala Thr Arg225 230 235
240Gln Met Leu Ile Gly Thr Val Leu His Glu Val Phe Gln Lys Ala
Ile 245 250 255Asn Asn Ser
Phe Ala Pro Glu Lys Leu Gln Glu Leu Ala Phe Gln Thr 260
265 270Ile Gln Glu Ile Arg His Leu Lys Glu Met
Tyr Arg Leu Asn Leu Ser 275 280
285Gln Asp Glu Ile Lys Gln Glu Val Glu Asp Tyr Leu Pro Ser Phe Cys 290
295 300Lys Trp Ala Gly Asp Phe Met His
Lys Asn Thr Ser Thr Asp Phe Pro305 310
315 320Gln Met Gln Leu Ser Leu Pro Ser Asp Asn Ser Lys
Asp Asn Ser Thr 325 330
335Cys Asn Ile Glu Val Val Lys Pro Met Asp Ile Glu Glu Ser Ile Trp
340 345 350Ser Pro Arg Phe Gly Leu
Lys Gly Lys Ile Asp Val Thr Val Gly Val 355 360
365Lys Ile His Arg Gly Tyr Lys Thr Lys Tyr Lys Ile Met Pro
Leu Glu 370 375 380Leu Lys Thr Gly Lys
Glu Ser Asn Ser Ile Glu His Arg Ser Gln Val385 390
395 400Val Leu Tyr Thr Leu Leu Ser Gln Glu Arg
Arg Ala Asp Pro Glu Ala 405 410
415Gly Leu Leu Leu Tyr Leu Lys Thr Gly Gln Met Tyr Pro Val Pro Ala
420 425 430Asn His Leu Asp Lys
Arg Glu Leu Leu Lys Leu Arg Asn Gln Met Ala 435
440 445Phe Ser Leu Phe His Arg Ile Ser Lys Ser Ala Thr
Arg Gln Lys Thr 450 455 460Gln Leu Ala
Ser Leu Pro Gln Ile Ile Glu Glu Glu Lys Thr Cys Lys465
470 475 480Tyr Cys Ser Gln Ile Gly Asn
Cys Ala Leu Tyr Ser Arg Ala Val Glu 485
490 495Gln Gln Met Asp Cys Ser Ser Val Pro Ile Val Met
Leu Pro Lys Ile 500 505 510Glu
Glu Glu Thr Gln His Leu Lys Gln Thr His Leu Glu Tyr Phe Ser 515
520 525Leu Trp Cys Leu Met Leu Thr Leu Glu
Ser Gln Ser Lys Asp Asn Lys 530 535
540Lys Asn His Gln Asn Ile Trp Leu Met Pro Ala Ser Glu Met Glu Lys545
550 555 560Ser Gly Ser Cys
Ile Gly Asn Leu Ile Arg Met Glu His Val Lys Ile 565
570 575Val Cys Asp Gly Gln Tyr Leu His Asn Phe
Gln Cys Lys His Gly Ala 580 585
590Ile Pro Val Thr Asn Leu Met Ala Gly Asp Arg Val Ile Val Ser Gly
595 600 605Glu Glu Arg Ser Leu Phe Ala
Leu Ser Arg Gly Tyr Val Lys Glu Ile 610 615
620Asn Met Thr Thr Val Thr Cys Leu Leu Asp Arg Asn Leu Ser Val
Leu625 630 635 640Pro Glu
Ser Thr Leu Phe Arg Leu Asp Gln Glu Glu Lys Asn Cys Asp
645 650 655Ile Asp Thr Pro Leu Gly Asn
Leu Ser Lys Leu Met Glu Asn Thr Phe 660 665
670Val Ser Lys Lys Leu Arg Asp Leu Ile Ile Asp Phe Arg Glu
Pro Gln 675 680 685Phe Ile Ser Tyr
Leu Ser Ser Val Leu Pro His Asp Ala Lys Asp Thr 690
695 700Val Ala Cys Ile Leu Lys Gly Leu Asn Lys Pro Gln
Arg Gln Ala Met705 710 715
720Lys Lys Val Leu Leu Ser Lys Asp Tyr Thr Leu Ile Val Gly Met Pro
725 730 735Gly Thr Gly Lys Thr
Thr Thr Ile Cys Thr Leu Val Arg Ile Leu Tyr 740
745 750Ala Cys Gly Phe Ser Val Leu Leu Thr Ser Tyr Thr
His Ser Ala Val 755 760 765Asp Asn
Ile Leu Leu Lys Leu Ala Lys Phe Lys Ile Gly Phe Leu Arg 770
775 780Leu Gly Gln Ile Gln Lys Val His Pro Ala Ile
Gln Gln Phe Thr Glu785 790 795
800Gln Glu Ile Cys Arg Ser Lys Ser Ile Lys Ser Leu Ala Leu Leu Glu
805 810 815Glu Leu Tyr Asn
Ser Gln Leu Ile Val Ala Thr Thr Cys Met Gly Ile 820
825 830Asn His Pro Ile Phe Ser Arg Lys Ile Phe Asp
Phe Cys Ile Val Asp 835 840 845Glu
Ala Ser Gln Ile Ser Gln Pro Ile Cys Leu Gly Pro Leu Phe Phe 850
855 860Ser Arg Arg Phe Val Leu Val Gly Asp His
Gln Gln Leu Pro Pro Leu865 870 875
880Val Leu Asn Arg Glu Ala Arg Ala Leu Gly Met Ser Glu Ser Leu
Phe 885 890 895Lys Arg Leu
Glu Gln Asn Lys Ser Ala Val Val Gln Leu Thr Val Gln 900
905 910Tyr Arg Met Asn Ser Lys Ile Met Ser Leu
Ser Asn Lys Leu Thr Tyr 915 920
925Glu Gly Lys Leu Glu Cys Gly Ser Asp Lys Val Ala Asn Ala Val Ile 930
935 940Asn Leu Arg His Phe Lys Asp Val
Lys Leu Glu Leu Glu Phe Tyr Ala945 950
955 960Asp Tyr Ser Asp Asn Pro Trp Leu Met Gly Val Phe
Glu Pro Asn Asn 965 970
975Pro Val Cys Phe Leu Asn Thr Asp Lys Val Pro Ala Pro Glu Gln Val
980 985 990Glu Lys Gly Gly Val Ser
Asn Val Thr Glu Ala Lys Leu Ile Val Phe 995 1000
1005Leu Thr Ser Ile Phe Val Lys Ala Gly Cys Ser Pro
Ser Asp Ile 1010 1015 1020Gly Ile Ile
Ala Pro Tyr Arg Gln Gln Leu Lys Ile Ile Asn Asp 1025
1030 1035Leu Leu Ala Arg Ser Ile Gly Met Val Glu Val
Asn Thr Val Asp 1040 1045 1050Lys Tyr
Gln Gly Arg Asp Lys Ser Ile Val Leu Val Ser Phe Val 1055
1060 1065Arg Ser Asn Lys Asp Gly Thr Val Gly Glu
Leu Leu Lys Asp Trp 1070 1075 1080Arg
Arg Leu Asn Val Ala Ile Thr Arg Ala Lys His Lys Leu Ile 1085
1090 1095Leu Leu Gly Cys Val Pro Ser Leu Asn
Cys Tyr Pro Pro Leu Glu 1100 1105
1110Lys Leu Leu Asn His Leu Asn Ser Glu Lys Leu Ile Ile Asp Leu
1115 1120 1125Pro Ser Arg Glu His Glu
Ser Leu Cys His Ile Leu Gly Asp Phe 1130 1135
1140Gln Arg Glu 11451113DNAHomo sapiensmisc_feature(6)..(8)n
is a, c, g, or t 11ggtcannntg acc
131220DNAArtificial sequenceCDC2 forward primer for PCR
reaction 12cccaaatgga aaccaggaag
201320DNAArtificial sequenceCDC2 reverse primer for PCR reaction
13cgtttggctg gatcatagat
201420DNAArtificial sequenceCDC6 forward primer for PCR reaction
14ggaaacccgt ttgacaaagg
201524DNAArtificial sequenceCDC6 reverse primer for PCR reaction
15cgggtggggt gtaagagaag aatt
241620DNAArtificial sequenceCKS2 forward primer for PCR reaction
16ctgatgtctg aagaggagtg
201720DNAArtificial sequenceCKS2 reverse primer for PCR reaction
17ggaagaggtc gtctaaagag
201820DNAArtificial sequenceDNA2L forward primer for PCR reaction
18ggatggaact gttggtgaac
201920DNAArtificial sequenceDNA2L reverse primer for PCR reaction
19atttagtgag ggcacacacc
202018DNAArtificial sequenceERbeta forward primer for PCR reaction
20tccatgcgcc tggctaac
182122DNAArtificial sequenceERbeta reverse primer for PCR reaction
21cagatgttcc atgcccttgt ta
222224DNAArtificial sequenceBeta actin forward primer for PCR reaction
22acccacactg tgcccatcta cgag
242326DNAArtificial sequenceBeta actin reverse primer for PCR reaction
23tctccttaat gtcacgcacg atttcc
26
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